Means and methods for mediating protein interference

ABSTRACT

The present invention belongs to the field of functional proteomics and more particularly to the field of protein aggregation. The invention discloses a method for interfering with the function of a target protein and uses a non-naturally, user-designed molecule, designated as interferor, that has a specificity for a target protein and which induces aggregation upon contact with said target protein. The present invention also discloses such interferor molecules and their use in agrobiotech applications.

FIELD OF THE INVENTION

The present invention belongs to the field of functional proteomics andmore particularly to the field of protein aggregation. The inventiondiscloses a method for interfering with the function of a target proteinand uses a non-naturally, user-designed molecule, designated asinterferor, that has a specificity for a target protein and whichinduces aggregation upon contact with said target protein. The presentinvention also discloses such interferor molecules and their use inagrobiotech applications.

BACKGROUND OF THE INVENTION

Biology is entering an exciting era brought about by the increase ingenome-wide information. As genome sequencing and high-throughputfunctional genomics approaches generate more and more data, researchersneed new ways to tease out biological relevant information. Functionalgenomics in particular is making rapid progress in assigning biologicalmeaning to genomic data. The information encoded in the genome comprisesgenes, the protein products of which mediate most of the functions inorganisms, and control elements. Proteins were thought to be the mostimportant effectors in the cells, although recently non-coding RNAs havealso been identified as important players in regulatory processes.

Several key biological questions are central to continuing genomeprojects and are relevant to any cellular organism, from bacteria tohumans. One challenge is to understand how genes that are encoded in agenome operate and interact to produce a complex living system. Arelated challenge is to determine the function of all the sequenceelements in the genome. The toolbox of functional genomics has enabledseveral systematic approaches that can provide the answers to a fewbasic questions for the majority of genes in a genome, including when isa gene expressed, where its product is localized, with which other geneproducts does it interact and what phenotype results if a gene ismutated. Phenotypic analysis of mutants has been a powerful approach fordetermining gene function. Gene function can be altered through genedeletions, insertional mutagenesis and RNA interference (RNAi). RNAi isa relatively recent development for reducing gene expression. It followsreports of gene silencing in plants and other model organisms, and isbased on the observation from C. elegans that adding double-stranded RNA(dsRNA) to cells often interferes with gene function in asequence-specific manner. In many cases, the level of functionalreduction cannot be adequately controlled, is incomplete, the level ofspecificity is not entirely predictable and in some organisms RNAi doesnot work (e.g. in the yeast Candida albicans).

It is obvious that functional genomics has changed the way biology isdone and yet the field is still in its infancy in terms of detailing thecomplexity that underlies biological systems, such as the complexnetwork of genetic regulation, protein interactions and biochemicalreactions that make up a cell. Clearly there is a need to developinnovative technologies, especially in the field of functionalproteomics, in order to accelerate discoveries and to maximize thepotential offered by complementary methods in functional genomics. Itwould be desirable to possess a flexible technology that can directlytarget the biological function of a particular extracellular orintracellular protein instead of targeting the mRNA that translates itor manipulating the gene that encodes it.

The conversion of normally soluble proteins into conformationallyaltered insoluble proteins is thought to be a causative process in avariety of diseases such as for example the occurrence of amyloid betapeptide in Alzheimer's disease and cerebral amyloid angiopathy,alpha-synuclein deposits in Lewy bodies of Parkinson's disease, prionsin Creutzfeldt-Jacob disease, superoxide dismutase in amyotrophiclateral sclerosis and tau in neurofibrillary tangles in frontal temporaldementia and Pick's disease. Thus far, protein aggregation has mainlybeen studied as an unwanted, disease-causing phenomenon and it is widelyaccepted that cross-beta mediated aggregation is the most frequentlyoccurring and biologically relevant mechanism of aggregation².Cross-beta aggregation is the term used to indicate that aggregation isnucleated via the formation of intermolecular beta-sheets to which eachmolecule in the aggregate contributes an identical strand of typicallycomprising at least three contiguous amino acids. There is now abundantdata to show that the individual strands interact to form anintermolecular beta sheet and that this structure forms the backbone ofthe aggregate^(3,4). Self-association regions in target proteins can bedetermined by computer programs, such as TANGO⁶, which were developedfor predicting the aggregation propensity of peptides and proteins. Onespecific form of aggregation, namely the highly ordered amyloid fibre,is already being explored in the art for potential use in the materialsciences⁵. In addition, WO03102187 (Scegen, Pty Ltd) discloses a methodfor enhancing the activity of a molecule by fusing said molecule with amembrane translocating sequence, whereby the resulting chimeric moleculeself-assembles into a higher molecular weight aggregate. US20050026165(Areté Associates) discloses the use of conformational peptides, able tointeract with the beta-sheet conformation of insoluble proteins such asprions, as a diagnostic tool for prion diseases.

SUMMARY OF THE INVENTION

The present invention relates to a technology for the controlled andinducible protein aggregation of specific target proteins. The inventionalso provides de novo designed molecules, herein designated asinterferor molecules, which comprise at least one aggregation region ofwhich said aggregation region is derived from a target protein. In apreferred embodiment the interferor molecule comprises at least oneself-association region that is fused to a moiety that preventsaggregation of said self-association region. Upon contact between achosen target protein and a specifically designed interferor molecule, aspecific co-aggregation occurs between the target and the interferorresulting in a functional knock-out or a down-regulation of thebiological function for said target protein. This protein knock-down isconditional upon the presence of aggregates, which are induced by thepresence of the interferor molecule. An additional advantage is that thestrength of the protein interference can be experimentally controlled byvarying the number of aggregation regions in the interferor molecule.The invention does not only provide an efficient tool to down-regulatethe biological function of a specific extra- or intracellular proteinbut has also important therapeutic, agricultural and diagnosticapplications.

In a first aspect the invention provides for a method fordown-regulating the biological function of a protein in a plant or plantcell, the method comprising contacting said protein with a non-naturallyoccurring molecule, wherein said molecule comprises at least onebeta-aggregation region derived from said target protein.

In a specific aspect the beta-aggregating region in the non-naturallyoccurring molecule is fused to a moiety that prevents aggregation ofsaid β-aggregating region.

In a further specific aspect said moiety is a peptide or a proteindomain.

In a further specific aspect the beta-aggregating region consists of atleast 3 contiguous amino acids.

In yet a further specific aspect a polypeptide linker is present betweenthe beta-aggregating region and a moiety that prevents aggregation ofsaid β-aggregating region.

In yet a further specific aspect the non-naturally occurring molecule isa polypeptide encoded by a nucleotide sequence present on a recombinantvector and which, upon introduction into a plant cell or plant, producessaid polypeptide in said plant cell or plant.

In yet a further specific aspect the invention provides for anartificial gene encoding a non-naturally occurring polypeptide whereinsaid polypeptide is comprises at least one beta-aggregation regionisolated from a target protein.

In yet a further specific aspect the at least one beta-aggregationregion encoded by the artificial gene consists of at least 3 contiguousamino acids.

In yet a further specific aspect the at least one beta-aggregationregion encoded by the artificial gene is fused to a moiety that preventsaggregation of said beta-aggregation region.

In yet a further specific aspect the invention provides for arecombinant vector comprising an artificial gene encoding anon-naturally occurring polypeptide wherein said polypeptide comprisesat least one beta-aggregation region isolated from a target protein.

In yet another specific aspect the invention provides for a plant orplant cell or plant seed comprising an artificial gene as describedbefore or comprising a recombinant vector as described herein before.

FIGURE LEGENDS

FIG. 1. (a) TANGO plot diagram for BIN2; the peaks represent the peptidesequences with the highest propensity to aggregate within the BIN2protein. (b) Schematic representation of the bait249 expression vectorscontaining a booster of aggregation N-terminally fused to GFP. (c)Representation of bait249 expression vectors including different linkerand flanking sequences (aa sequences are indicated) but not containingany booster of aggregation.

FIG. 2. (a-e) CLSM evaluation of aggregates formation in N. benthamianaagro-infiltrated leaves transiently transformed with the GFP expressingconstructs indicated above each panel. Epidermal cells are GFP positivebut show different localization patterns mainly in the perinuclear area.White arrow indicates an insoluble inclusion body. Size bars: 10 μm.

FIG. 3. Upper panel: CLSM images of N. benthamiana epidermal cells after4.5 days from co-injection with 35SBIN2GFP and pMDCbait249NF_Tandexpressing strains. In the bottom panel: corresponding imagesrepresenting co-localization quantification performed by ImageJ MBFsoftware. Mender's overlap coefficients (0<R<1) for each picture areindicated; size bars represent 50 μm.

FIG. 4. Co-immunoprecipitation of 35S::bait249-GFPvariants co-expressedin N. benthamiana with 35S::BIN2:HA. In the left panel the Western blotdetection of the unbound fractions of the plant proteins extracts after4 hours of incubation with anti-GFP beads and detection with anti-HAantibody (left upper panel) or with anti-GFP antibodies (left lowpanel). In the right panel the detection of the Immuno Precipitated (IP)beads with anti-HA (right upper panel) or with anti-GFP antibody (rightlower panel).

FIG. 5. (a-d) CLSM images of Arabidopsis 8 D.A.S. T3 seedlingsexpressing 35S::bait249R-GFP construct. Epidermal cells in cotyledons(a), hypocotyl (b) and root cells (c) show perinuclear aggregation(white arrows). The root tip shows no clear cytosolic aggregation andweak GFP signal (d). (e-h) CLSM images of Arabidopsis 8 D.A.S. T3seedlings expressing 35S::bait249NF_Tand-GFP construct. Epidermal cellsin cotyledons (e), hypocotyl (f) and root cells (g) show cytosolicaggregation. The root tip shows weaker GFP intensity (h). Size bars areindicated.

FIG. 6. (a-d) CLSM images of Arabidopsis 8 D.A.S. T3 seedlingsexpressing 35S::bait249-GFP construct. No clear aggregation is visiblein plant tissues but only weak GFP expression is visible in cells incotyledons (a), hypocotyls (b), and root tip (d). In root cells (c) thepresence of insoluble aggregates in the form of round-shaped bodies isevidenced (white arrow). (e-h) CLSM images of Arabidopsis 8 D.A.S. T3seedlings expressing 35S::bait249NF-GFP construct. The bait is expressedin any plant tissue showing perinuclear aggregation only in root cells(g). Size bars are indicated.

FIG. 7. TEM evaluation of immunogold labelled ultrathin section of 8D.A.S. Arabidopsis seedlings incubated with an anti-GFP antibody. (a)Hypocotyl vascular parenchyma cell expressing 35S::bait249R-GFP showinglabeled cytosolic fibrillar material (a) enlarged in the inset. (b-c)Details of root elongation area cells showing clustered labeling ofbait249NF_Tand-GFP in the cytosol (b) and close to a Golgi stack (c).(d) Cotyledon palisade cell expressing bait249NF_Tand-GFP evidencingperinuclear labeling, enlarged in the inset. (e) Root elongation areacell showing cytoplasmic labeling of bait249NF_Tand in the cytosol. Sizebars are indicated.

FIG. 8. (a) Native-PAGE and anti-GFP detection of high molecular weightcomplexes (framed) in protein extracts from transgenic Arabidopsisplants stably expressing the BIN2 bait249 lines respect to wild type(Col-0) plant extracts. (b,c) FT-IR spectroscopy on immuno-precipitatedmaterial from transgenic plants expressing 35S::bait249-GFP,35S::bait249R-GFP, 35S::bait249NF_Tand-GFP and 35S::bait249NF-GFP. Theincreased absorbance at 1616 and at 1680 (black arrows) values indicatethe presence of β-sheet aggregates.

FIG. 9. (a) Phenotype of 35S::bait249R-GFP and 35S::bait249NF_Tand-GFPArabidopsis seedlings compared to Col-0 grown vertically in vitro for 8days under long day photoperiod and in soil for 1.5 months.Quantification of roots and hypocotyls lengths on an average of 50 8D.A.S. seedlings per line is also represented. (b) Brazzinazoleresistance dose response assay for 35S::bait249R-GFP and35S::bait249NF_Tand-GFP Arabidopsis 4 D.A.S. seedlings lines compared toCol-0 and triple GSKs group II T-DNA mutant (trGSKsII_k.o.).Corresponding quantification of hypocotyls lengths on an average of 50seedlings per line is represented in the graph.

FIG. 10 a) Relative expression levels of the BR-biosynthetic genes DWF4and CPD and of the gene for the BR-responsive NAC transcription factor(At5g46590) in 8 D.O. Arabidopsis seedlings grown in vitro under longday conditions. b) Chaperone genes (HSP70, HSP90-1, HSP101, HSC70-1,HSC70-2 and HSC70-3) expression levels measured in the same experimentalconditions. In each case the mRNA amount was normalized to the level ofCDKA1 as reference gene.

FIG. 11. TEM ultrastructural evaluation of 35S::bait249R-GFPcytotoxicity in Arabidopsis plants. A low magnification comparisonbetween hypocotyls and root cells in Col-0 (a,c) and the mutant (e,g) isshowed. High magnification micrographs of the same areas in Col-0 (b,d)and mutant (f,h). Size bars are indicated.

FIG. 12. Upper panel: CLSM images of 8 D.A.S. Arabidopsis seedlingsexpressing 35S::BIN2-GFP and pMDC::bait249NF_Tand_RFP after 24 hours ofinduction. In the bottom panel: corresponding images representingco-localization quantification performed by ImageJ MBF software.Mander's overlap coefficients (0<R<1) for each picture are indicated.

AIMS AND DETAILED DESCRIPTION OF THE INVENTION

This application is a continuation in part of copending application Ser.No. U.S. Ser. No. 12/214,761 filed Jun. 20, 2008 (published asUS20090012275).

In the present invention we have developed a process for down-regulatingthe biological function of a protein through the use of interferormolecules that have a specificity for a target protein. Upon contactwith a target protein a co-aggregation occurs between the interferormolecule and the target. The aggregation withdraws the target from itssoluble environment and results in a functional knock-down of the targetprotein.

Thus in one embodiment the invention provides a method fordown-regulating the biological function of a protein comprisingcontacting said protein with a non-naturally occurring moleculecomprising at least one self-association region isolated from saidprotein.

In another embodiment the invention provides a method fordown-regulating the biological function of a protein comprisingcontacting said protein with a non-naturally occurring moleculeconsisting of at least one self-association region isolated from saidprotein.

In yet another embodiment the invention provides a method fordown-regulating the biological function of a protein comprisingcontacting said protein with a non-naturally occurring moleculecomprising at least one self-association region isolated from saidprotein wherein said self-association domain is fused to a moiety thatprevents aggregation of said self-association region.

In yet another embodiment the invention provides a method fordown-regulating the biological function of a protein comprisingcontacting said protein with a non-naturally occurring moleculeconsisting of at least one self-association region isolated from saidprotein wherein said self-association domain is fused to a moiety thatprevents aggregation of said self-association region.

In yet another embodiment the invention provides a method fordown-regulating the biological function of a protein in a plant or plantcell, the method comprising: i) contacting said protein with anon-naturally occurring molecule, wherein said protein comprises a firstβ-aggregating region, said non-naturally occurring molecule comprises asecond β-aggregating region, and said first and second β-aggregatingregions are identical, ii) intermolecular beta-aggregation occursbetween the protein and the non-naturally occurring molecule, iii) thenon-naturally occurring molecule is a polypeptide, and iv) saidbiological function is down-regulated, wherein the contacting betweenthe protein and the non-naturally occurring molecule is produced byexpression of said non-naturally occurring molecule in said plant orplant cell.

In yet another embodiment the invention provides a method fordown-regulating the biological function of a protein comprisingcontacting said protein with a non-naturally occurring molecule whichcomprises part A and part B wherein i) part A is a peptide, or a proteindomain or an agarose bead preventing aggregation of part B and ii) partB which comprises at least 1 self-association region consisting of atleast 3 contiguous amino acids and wherein said region is isolated fromsaid protein which function is to be down-regulated with, and wherein alinker is optionally present between parts A and B.

In yet another embodiment the invention provides a method fordown-regulating the function of a protein comprising contacting saidprotein with a non-naturally occurring molecule which comprises part Aand part B wherein i) part A is a peptide, or a protein domain or anagarose bead preventing aggregation of part B so that part B is indirect contact with the solvent wherein said molecule and said proteinare present and ii) part B which comprises at least 1 self-associationregion wherein said region consists of at least 3 contiguous amino acidsand wherein said region is isolated from said protein which function isto be down-regulated with, and wherein a linker is optionally presentbetween parts A and B.

In particular embodiments the self association region (orbeta-aggregation region) consists of at least 4, at least 5, at least 6,at least 7, at least 8 or at least 9 contiguous amino acids wherein saidregion is isolated from a target protein.

In another embodiment part B of the non-naturally occurring moleculecomprises at least 2 self-association regions wherein at least one ofsaid regions is derived from said protein which function is to beinterfered with.

The term ‘non-naturally occurring molecule’ refers to the fact that suchan interferor molecule is man made. For instance, when an interferormolecule is polypeptide (id est both part A and B are peptides) suchpolypeptide is designed by isolating part B from a target protein (idest the self association region) and by coupling said part B to a part Awhich can be derived (i) from another protein or (ii) from the sametarget protein in which case said part A is not present immediatelyadjacent to part B. In still other words the self-association regionderived from the target fused to a moiety (when the interferor is apolypeptide said moiety is also a polypeptide) that prevents theaggregation of the self-association region is different from a naturallyoccurring fusion between part A and B by at least one natural aminoacid. Typically, such interferor molecule will not exist as a contiguouspolypeptide in a protein encoded by a gene in a non-recombinant genome.In the present invention it is understood that the ‘non-naturallyoccurring molecule’ or more specifically the ‘non-naturally occurringpolypeptide’ can be encoded by an ‘artificial gene’.

It should be clear that interferor molecules can be designed in amodular fashion, by introducing repetition and changing the order of theparts A and B. A non-limiting list of the following combinations is: aninterferor with the A-B-structure, an interferor with the B-A-structure,an interferor with the A-B-A-structure, an interferor with theB-A-B-structure, an interferor with the A′-B-A″ structure and aninterferor with the B′-A-B″ structure wherein a linker (spacer) isoptionally present between parts A, A′, A″ and B, B′, B″. A, A′ and A″are different of similar moieties (e.g. different peptide sequences). B,B′ and B″ are different or similar self association sequences (e.g. B isa self-association sequence derived from the target protein and B′ is asynthetic self-association sequence). In the context of the presentinvention a ‘self association region’ is herein equivalent with thewording a ‘beta-aggregation region’.

In still other words the invention provides a method for down-regulatingthe biological function of a protein comprising contacting said proteinwith a molecule comprising at least one self-association region isolatedfrom said protein wherein said self-association region is fused to amoiety that prevents aggregation of said self-association region so thatsaid self-association region is in direct contact with the solventwherein said molecule and said protein are present. From the above itshould be clear that said ‘moiety’ is equivalent with the term part Aand part B is equivalent with the wording ‘at least one self associationregion’.

The wording ‘down-regulating the function of a protein’ means that thenormal biological activity of a protein is reduced (inhibited,down-regulated, reduced and disrupted are equivalent words here) or thatthe protein is withdrawn from its normal biological environment (e.g. aprotein which is a normal resident of the endoplasmic reticulum is notpresent through down-regulation of its function). Thus, by applying themethod of the invention the function of a protein is disrupted throughan aggregation of said protein by contacting said protein with thenon-natural molecule of the present invention. Said non-natural moleculeis herein designated as ‘the interferor’ or the ‘interferor molecule’.Aggregation refers to the fact that a protein which is normally solubleis changed into an insoluble protein or an aggregated protein in itsnormal biological environment through direct contact or binding with theinterferor. The wording ‘down-regulating the function of a protein’ canalso be interchanged by the wording ‘knocking down the function of aprotein’ or ‘negatively interfering with the function of a protein’. Thedown-regulation of the function of a protein can also mean that aprotein is not present anymore in a soluble form in the cell or that aprotein is not present anymore in a soluble form in its normalbiological environment (e.g. (sub)-cellular or extra-cellularlocalization). In addition, it can also mean that the aggregated proteinis degraded through the natural clearance mechanisms of the cell and isno longer detectable in soluble or insoluble form. In addition, it canalso mean that a transmembrane receptor protein cannot bind its normalligand anymore through interferor induced aggregation of saidtransmembrane protein. Thus the down-regulation of the function of aprotein can also mean that a protein which is a normal resident of e.g.the mitochondria is not present there anymore through the method ofprotein interference. In a particular embodiment the ‘down-regulation ofthe function of a protein’ or ‘the negative interference with thefunction of a protein’ or ‘knocking down the function of a protein’ isat least a 20%, at least a 30%, at least a 40%, at least a 50%, at leasta 60%, at least a 70%, at least a 80%, at least a 90%, at least a 95% oreven a 100% loss of function as compared to the normal (100%) functionof the protein.

The function of a protein or the lack of presence of a protein in itsnormal biological environment (localization) can conveniently bedetermined by methods known in the art. For example, depending on thetarget protein of interest, the function can be determined by measuringthe reduced enzymatic activity. The reduced presence of a protein in itsnormal biological localization can for example be measured by the lackof formation of a complex, the lack of the occurrence of a targetprotein in a sub-cellular compartment, the presence of the targetprotein in soluble form, the presence of the target protein in anaggregated (insoluble is an equivalent term here) form. Alternatively,the effect of the down-regulation of a target protein can be measured ina cellular assay (e.g. loss or gain of growth, loss or gain of invasion,loss or gain of proteolytic activity).

In a particular embodiment such normal biological activity (or normalfunction or normal localization) of a protein can be interfered withintracellularly or extracellularly. ‘Intracellularly’ refers to thelocalization of a protein inside the cell of an organism or host (e.g.the cytoplasm, the mitochondria, the lysosome, the vacuole, the nucleus,the chloroplast, the endoplasmic reticulum (ER), the cellular membrane,the mitochondrial membrane, the chloroplast membrane, . . . ).‘Extracellularly’ not only refers to the localization of a protein inthe extracellular medium of the cell but also refers to proteins whichcontact the extracellular medium such as a membrane-anchored proteins, atransmembrane protein etc. Non-limiting examples of extracellularproteins are secreted proteins (e.g. proteases, antibodies and cytokinespresent in the blood or plasma) or proteins present in the extracellularmatrix (e.g. matrix metalloproteins and transmembrane proteins (e.g. agrowth factor receptor)).

Cells or hosts which can be targeted with the method of the inventioncomprise prokaryotic and eukaryotic cells. Non-limiting examples areviruses, bacteria, yeasts, fungi, protozoa, plants and mammals includinghumans.

It should be clear that the method of down-regulation the biologicalfunction of a protein can be used to interfere with the biologicalfunction with 1, 2, 3, 4, 5 or even more proteins simultaneously.Particularly since part B comprises at least one self-associationregion, part B can for example comprise different self-associationregions each specific for a different protein. The interferor used forinterference with the biological function of at least one target proteinis not naturally present in nature and can be made through chemicalsynthesis or through recombinant protein expression or through acombination of the latter.

Thus an interferor molecule comprises at least one self-associationregion (thus part B comprises at least one self-association region). A‘self-association region’ is herein defined as a contiguous sequence ofamino acids that has a high tendency to form a tight molecular assemblywith identical or very closely related sequences. The wording ‘has ahigh tendency to form a tight molecular assembly’ can also be construedas ‘has a high affinity’. Affinity is usually translated into values ofdissociation (Kd-values). Kd-values between interferor and targetproteins are typically lying between micromolar and nanomolar ranges,but can be sub-nanomolar or supra-micromolar. Examples ofself-association regions are intermolecular beta sheet regions,alpha-helical elements, hairpin loops, transmembrane sequences andsignal sequences. In a particular embodiment at least oneself-association region is present in part B. In another particularembodiment at least two self-association regions are present in part B.In another particular embodiment 3, 4, 5, 6 or more self-associationregions are present in part B.

Said self-association regions can be interconnected by a linker region(e.g. a spacer of about 2 to about 4 amino acids). One (or at least one)self-association region present in part B is derived from a targetprotein. In a particular embodiment 2, 3, 4, 5, 6 or moreself-association regions in part B are derived from a target protein. Inanother particular embodiment 2, 3, 4, 5, 6 or more self-associationregions in part B are derived from more than 1 target protein. Inanother particular embodiment the at least two self-association regionspresent in part B are derived from the same target protein. The targetprotein is defined herein as the protein with which one wants tointerfere with its function. Thus, in order to make part B specific forat least one protein at least one self-association region in part Bshould be ‘derived from’ the target protein or at least oneself-association region should be present in said target protein.‘Derived from’ means that at least one contiguous self-associatingregion should be identical or homologous in amino acid sequence to acontiguous region of said target protein. In a preferred embodiment,said at least one self-associating region is at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or at least 100% identical to the self-association regionpresent in said target protein region.

It is preferred that the length of a self-association region consists ofat least 3 contiguous amino acids. In a preferred embodiment said regionconsists of about 3 to about 30 amino acids. In another preferredembodiment said region consists of about 3 to about 25 amino acids. In aparticularly preferred embodiment said region consists of about 5 toabout 20 amino acids.

Self-association regions present in part B of the interferor moleculealso can be determined and isolated from proteins other than the targetprotein and said self-association regions are coupled with at least oneself-association region derived from the target protein, optionally witha spacer (or linker) between said self-association regions. For example,self-association regions that can be used can be derived fromself-association regions of proteins which do not normally occur in thehost in which the down-regulation of the biological function of a targetprotein is performed (thus some self-association regions in part B canbe taken from an unrelated organism). The nature of the self-associationregions determine the level of inhibition (id est the strength ofinhibition) of a target protein through induced aggregation. More thanone self-association region can be used from a target protein in aninterferor molecule but also synthetic self-association regions orself-association regions derived from a different target protein can beused in combination with one or more self-association regions from atarget protein.

In a particular embodiment such self-association regions consist of asynthetic sequence which is not derived from existing proteins and hencedoes not occur in nature. Examples of such synthetic self associationregions are described in Lopez de la Paz M. et al (2002) PNAS 99, 25, p.16053, table 1 which is herein incorporated by reference.

If at least one self-association region (id est the part B of theinterferor molecule) has a hydrophobic character (because of itsaggregation inducing properties) it is preferably fused (or linked orcoupled which are equivalent terms) to a moiety (id est part A of theinterferor molecule) that prevents aggregation of said self-associationregion and exposes said self-association region in direct contact withthe solvent in which the interferor is present. As such, in certainembodiments part A has a solubilizing function to keep part B insolution. In such embodiments said part A is for example a peptide, aprotein domain, a protein (preferably different from the target protein,see example 2), a glycosylation structure, a (hydrophilic) chemicalgroup or a cyclodextrine or derivative thereof. In certain otherembodiments said part A is an agarose bead, a latex bead, a cellulosebead, a magnetic bead, a silica bead, a polyacrylamide bead, amicrosphere, a glass bead or any solid support (e.g. polystyrene,plastic, nitrocellulose membrane, glass).

In the interferor molecules part B and part A may be optionally linked(or coupled) by means of a linker region (a spacer is an equivalentword). Said linker region can for instance be an unnatural linker madeby chemical synthesis (e.g. a flexible linker such as ahydroxy-substituted alkane chain, dextran, polyethylene glycol or thelinker can also consist of amino acid homologues) or said linker canexist of natural amino acids such as a poly(threonine) or poly(serine).Preferentially when the linker comprises amino acids, the length of saidlinker region is between about 3 and about 15 amino acids, morepreferably between about 5 and about 10 amino acids. Often a flexiblelinker can be chosen but it is envisaged that a stiff linker will alsowork. Flexible linker sequences can be taken from nature, mostly suchregions connect domains in naturally occurring proteins, such as thelinker between the SH2 and SH3 domains src tyrosine kinase or the linkerbetween the BRCT domains of BRCA1.

The term ‘contacting’ refers to the process in which the interferor andthe target protein interact. In one form the interferor is added (e.g.interferor is present at a particular concentration in a solution) to asample comprising the target protein. In another form the interferormolecule is injected into an organism comprising the target protein.Contacting can for example also be carried out through the process oftransformation of a cell comprising the target protein, e.g. an isolatedcell, e.g. in cell culture, a unicellular microorganism or a cell or aplurality of cells within a multicellular organism. Transformationimplies that the interferor molecule is introduced in a host (e.g. acell) through commonly known transfection or transformation methods(e.g. by gene transfer techniques including calcium phosphate,DEAE-dextran, electroporation, microinjection, viral methods, the use ofcationic liposomes (see for example Feigner, P. L. et al. (1987), Proc.Natl. Acad. Sci USA 84, 7413), commercially available cationic lipidformulations e.g. Tfx 50 (Promega) or Lipofectamin2000 (LifeTechnologies), particle bombardment, etc.). The interferor molecule maybe encoded by a recombinant vector (e.g. a plasmid, cosmid, viralvector) and can be synthesized inside a host. In an alternativeembodiment the interferor molecule can be introduced into a cell throughcarrier-mediated delivery, e.g. by liposomal carriers or nano-particlesor by injection. In yet another alternative embodiment the interferormolecule can enter a cell through a sequence which mediates cellpenetration (or cell translocation). In the latter case the interferormolecule is further modified through the recombinant or syntheticattachment of a cell penetration sequence. Thus, the interferor molecule(e.g. as a polypeptide) may be further fused or chemically coupled to asequence facilitating transduction of the fusion or chemical coupledproteins into prokaryotic or eukaryotic cells. Sequences facilitatingprotein transduction are known to the person skilled in the art andinclude, but are not limited to Protein Transduction Domains.Preferably, said sequence is selected from the group comprising of theHIV TAT protein, a polyarginine sequence, penetratin and pep-1. Stillother commonly used cell-permeable peptides (both natural and artificialpeptides) are disclosed in Joliot A. and Prochiantz A. (2004)Nature CellBiol. 6 (3) 189-193.

In a particular embodiment the interferor essentially consists of aminoacids. In some embodiments the sequences of parts A and B from theinterferor molecule are derived from the same target protein. In otherembodiments the interferor is a molecule meaning that the sequences fromparts A and B are derived from different proteins, e.g. part A isderived from one protein and at least one aggregation region of part Bis derived from the target protein. A “Polypeptide” refers to a polymerin which the monomers are amino acids and are joined together throughamide bonds, alternatively referred to as a peptide. When the aminoacids are alpha-amino acids, either the L-optical isomer or theD-optical isomer can be used. Additionally, unnatural amino acids, forexample, beta-alanine, phenylglycine and homoarginine are also included.Commonly encountered amino acids that are not gene-encoded may also beused in the present invention. All or part of the amino acids used inthe interferors may be either the D- or L-isomer. In addition, otherpeptidomimetics are also useful in the present invention. Wespecifically refer and incorporate herein the review of the developmentand use of peptidomimetics as antagonists for protein-proteininteractions from Sillerud L O and Larson R S (2005) Curr Protein PeptSci. 6(2):151-69. Furthermore, D-amino acids can be added to the peptidesequence to stabilize turn features (especially in the case of glycine).In another approach alpha, beta, gamma or delta turn mimics (such asalpha, beta, gamma, or delta di-peptides can be employed to mimicstructural motifs and turn features in a peptide and simultaneouslyprovide stability from proteolysis and enhance other properties such as,for example, conformational stability and solubility.

Isolation of a Self Association Region (or a Beta-Aggregation Region)from a Target Protein:

Self-association sequences are often hydrophobic but this is not alwaysthe case. For example, the self-associating regions of the yeast prionsare rather polar. In fact cross-beta aggregation of an amino acid regionderived from a polypeptide or protein can be initiated when (1) it has ahigh hydrophobicity, (2) it has a good β-sheet propensity, (3) it has alow net charge and (4) it is solvent-exposed. Thus, self-associationprotein regions (‘segment’ is an equivalent term for ‘region’) are mostoften buried in the folded state and are not exposed to the solvent. Thelatter is confirmed by the experimental finding that in many globularproteins, aggregation occurs during refolding or under conditions inwhich denatured or partially folded states are significantly populated,i.e. at high concentration or as a result of destabilizing conditions ormutations.

Based on these findings computer algorithms were developed that are ableto predict self-association regions (“β-aggregating stretches orsegments or regions” is an equivalent wording) in proteins. One suchalgorithm, TANGO, is based on a statistical mechanics algorithm thatconsiders the three physico-chemical parameters described above but alsoconsiders competition between different structural conformations:beta-turn, alpha-helix, beta-sheet aggregates and the folded state(Femandez-Escamilla, A M et al (2004) Nat. Biotechnol. 22, 1302-1306,especially the Methods section on pages 1305 and 1306 are hereinspecifically incorporated by reference and also the Supplementary Notes1 and 2 of the same article for further details on the methods and thedata sets used for the calibration and the testing of the TANGOalgorithm). Thus, self-association regions present in target proteinsare obtainable by computer algorithms such as TANGO. Self-associationregions are often buried inside the core of the target proteins¹⁰,effectively shielding the peptide from intermolecular association by anenergy barrier corresponding to the stability of the target proteins¹¹.In its normal environment (e.g. cytoplasm, extracellular matrix) thetarget protein has assistance from molecular chaperones that assist theprotein in keeping its functional, monomeric form¹². The model used bythe TANGO algorithm⁶ is designed to predict beta-aggregation in peptidesand proteins and consists of a phase-space encompassing the random coiland the native conformations as well as other major conformationalstates, namely beta-turn, alpha-helix and beta-aggregate. Every segmentof a peptide can populate each of these states according to a Boltzmanndistribution. Therefore, to predict self-association regions of apeptide, TANGO simply calculates the partition function of thephase-space. To estimate the aggregation tendency of a particular aminoacid sequence, the following assumptions are made: (i) in an orderedbeta-sheet aggregate, the main secondary structure is the beta-strand.(ii) the regions involved in the aggregation process are fully buried,thus paying full solvation costs and gains, full entropy and optimizingtheir H-bond potential (that is, the number of H-bonds made in theaggregate is related to the number of donor groups that are compensatedby acceptors. An excess of donors or acceptors remains unsatisfied).(iii) complementary charges in the selected window establish favorableelectrostatic interactions, and overall net charge of the peptide insidebut also outside the window disfavors aggregation. TANGO can be accessedon the World Wide Web at http://tango.embl.de/. The zyggregatoralgorithm is another example (Pawar A P et a/(2005) J. Mol. Biol. 350,379-392). These algorithms identify aggregation prone sequences bycomparing the aggregation propensity score of a given amino acidsequence with an average propensity calculated from a set of sequencesof similar length.

In the present invention we estimate that a self-association regionidentified within a target protein with a TANGO score of 5% correspondsto an aggregation risk in vitro of 95%⁶. We have calculated that 85% ofproteins from the human proteome that are not related to disease have atleast one region with a TANGO score above the experimentally determinedthreshold of 5%. This shows that although more than 85% of the humanproteins contain at least one single self-association region thataggregation is prevented because of the normal stability of the proteinand the assistance from the chaperone machinery. The present inventionisolates these self-association regions from target proteins for thepreparation of interferor molecules which are used for the specificinduction of protein aggregation. The B-part of the interferor moleculescomprises at least 1 aggregation region and at least one aggregationregion is derived from a target protein. It is possible to control thestrength of the protein interference (the strength of proteininterference is for example the % of loss of biological function of atarget protein when said protein or cell comprising said protein iscontacted with a specific interferor molecule) through the incorporationof more than one aggregation region of a target protein in the B-part ofthe interferor molecule. Indeed, aggregation regions derived from atarget protein with a low TANGO score (typically between 5% to about20%) can be repeated in the B-part of the interferor to 2, 3, 4 or moreaggregation regions. As an alternative embodiment 1, 2 or 3 or 4 or moredifferent aggregation regions with a low TANGO score derived from thesame protein can be incorporated into the B-part of the interferor. Asanother alternative embodiment 1, 2, 3, 4 or more synthetic aggregationregions (thus not derived from the target protein) can be combined with1, 2, 3, 4, or more aggregation regions derived from the target proteininto the B-part to enhance the down-regulation of a target protein witha low TANGO score.

Thus in another embodiment the invention provides a non-naturallyoccurring molecule capable of aggregating a target protein. In aparticular embodiment said non-naturally molecule is proteinaceous innature. Proteinaceous means that the molecule comprises L-amino acids orD-amino acids or a mixture of L- and D-amino acids or a combination ofnatural amino acids and peptidomimetics.

In yet another embodiment the invention provides a non-naturallyoccurring molecule comprising at least one self-association regionisolated from a protein domain capable of being soluble in water whereinsaid self-association region is fused to a moiety that preventsaggregation of said self-association region.

In yet another embodiment the invention provides a non-naturallyoccurring molecule comprising at least one self-association regionisolated from a protein domain capable of being soluble in water whereinsaid self-association region is fused to a moiety that preventsaggregation of said self-association region so that saidself-association region is in direct contact with the solvent wherein itis present.

In yet another embodiment the invention provides a non-naturallyoccurring molecule consisting of at least one self-association regionisolated from a protein domain capable of being soluble in water whereinsaid self-association region is fused to a moiety that preventsaggregation of said self-association region.

In yet another embodiment the invention provides a non-naturallyoccurring molecule consisting of at least one self-association regionisolated from a protein domain capable of being soluble in water whereinsaid self-association region is fused to a moiety that preventsaggregation of said self-association region so that saidself-association region is in direct contact with the solvent wherein itis present.

In a particular embodiment such a moiety is for example a peptide, anagarose bead, a protein domain or a protein. In another particularembodiment said non-naturally occurring molecule comprises at least twoself-association regions of which at least one self-association regionis derived from a target protein.

In other words the invention provides a non-naturally occurringmolecule, which comprises part A and part B wherein i) part A comprisesa region, such as a peptide, protein domain, protein or agarose beadpreventing the aggregation of part B, and ii) part B which comprises atleast 1 self-association region wherein said region consists of at least3 contiguous amino acids and wherein said region is isolated from saidprotein which function is to be interfered with, and wherein a linker isoptionally present between parts A and B.

In still other words the invention provides a non-naturally occurringmolecule which comprises part A and part B wherein i) part A comprises aregion, such as a peptide, protein domain or agarose bead preventing theaggregation of part B, and ii) part B which comprises at least 1self-association region consisting of at least 3 contiguous amino acidsand wherein at least one self-association region is isolated from aprotein which function is to be interfered with and wherein said regionis isolated from a domain from said protein which is capable of beingsoluble in water, and wherein a linker is optionally present betweenparts A and B, and wherein part B is in direct contact to theenvironment wherein said molecule and said protein are present.

In still other words the invention provides a non-naturally occurringmolecule which comprises part A and part B wherein i) part A comprises aregion, such as a peptide, protein domain or agarose bead preventing theaggregation of part B, and ii) part B which consists of at least 1self-association region consisting of at least 3 contiguous amino acidsand wherein said at least one self-association region is isolated from aprotein which function is to be interfered with and wherein said regionis derived from a domain from said protein which is capable of beingsoluble in water, and wherein a linker is optionally present betweenparts A and B, and wherein part B is in direct contact to theenvironment wherein said molecule and said protein are present.

In yet another embodiment the invention provides an artificial genecomprising the following operably linked DNA elements: a) a plantexpressible promoter b) a DNA region encoding for at least onebeta-aggregation region isolated from a target protein and c) a 3′ endregion comprising transcription termination and polyadenylation signalsfunctioning in cells of said plant.

In yet another embodiment the invention provides an artificial genecomprising the following operably linked DNA elements: a) a plantexpressible promoter b) a DNA region encoding for at least onebeta-aggregation region isolated from a target protein wherein saidbeta-aggregation region consists of at least 3 contiguous amino acidsand c) a 3′ end region comprising transcription termination andpolyadenylation signals functioning in cells of said plant.

In yet another embodiment the invention provides an artificial genecomprising the following operably linked DNA elements: a) a plantexpressible promoter b) a DNA region encoding for at least onebeta-aggregation region isolated from a target protein wherein saidbeta-aggregation region is fused to a moiety to prevent aggregation ofsaid beta-aggregation region and c) a 3′ end region comprisingtranscription termination and polyadenylation signals functioning incells of said plant.

The wording ‘isolated (or derived form) from a domain from said proteinwhich is capable of being soluble in water’ means that a selfassociation region is a contiguous amino acid sequence isolated from asoluble domain of a protein. The latter also means that self-associationregions derived from transmembrane regions or self-association regionsderived from signal sequences are specifically excluded in the claimscope of these interferor molecule products in such embodiments.

In the present invention the at least one self-association region of theinterferor molecule (id est part B of the interferor molecule), is ‘indirect contact’ with the environment (e.g. solvent, cytosol) in whichsaid interferor molecule is present. The importance of this is clarifiedfurther. In globular proteins self-association sequences (alsodesignated as ‘aggregation nucleating regions’) are generally buried inthe hydrophobic core of the globular protein and as such kept protectedfrom the solvent by a dense network of cooperative interactionsstabilizing the native state. Hence, under normal circumstances there isno ‘direct contact’ between said self-association region and theenvironment (for example the solvent). Only when the protein isunfolded, for example when it is synthesized on the ribosome ordestabilized by mutation, change of temperature, pH or loss of aspecific chaperone, thereby favoring the unfolded state, will it exposeits self-association regions to the environment. Self-associationregions are normally buried inside proteins (in order to preventaggregation) and in the non-natural interferor molecule saidself-association regions have been isolated and exposed to theenvironment by linking said regions to a moiety that preventsaggregation (id est part A of the interferor molecule). In still otherwords, the non-naturally interferor molecule does not fold into aglobular structure and therefore the at least one self-associationregion (id est part B) in the non-natural interferor molecule is indirect contact with the solvent in which said interferor molecule ispresent. Hence, ‘in direct contact’ refers to the opposite of ‘beingburied and kept protected from’.

In a specific embodiment the interferor molecules that comprise at leastone self-association region derived from a soluble protein domain arepolypeptides.

In another specific embodiment the invention provides a recombinantvector comprising a polynucleotide encoding such interferor molecules.

In another specific embodiment the interferor molecules of the inventionare used as a medicament.

In yet another embodiment the method of protein interference of theinvention may be used for determining the function of a protein in acell or an organism being capable of mediating protein interference. Thecell can be a prokaryotic cell or can be a eukaryotic cell or can be acell line, e.g. a plant cell or an animal cell, such as a mammaliancell, e.g. an embryonic cell, a pluripotent stem cell, a tumor cell,e.g. a teratocarcinoma cell or a virus-infected cell. The organism ispreferably a eukaryotic organism, e.g. a plant or an animal, such as amammal. In a particular embodiment a plant cell is used. In yet anotherparticular embodiment a plant protoplast is used.

The target protein to which the interferor molecule of the invention isdirected may be associated with a pathological condition. For example,the protein may be a pathogen-associated protein, e.g. a viral protein,a tumor-associated protein or an autoimmune disease-associated protein.The target protein may also be a heterologous gene expressed in arecombinant cell or a genetically altered organism. By inhibiting thefunction of such a protein valuable information and benefits in theagricultural field or in the medicine or veterinary medicine field maybe obtained. In a particularly preferred embodiment the method of theinvention is used with an eukaryotic cell or a eukaryotic non-humanorganism exhibiting a target protein-specific knockout phenotypecomprising an at least partially deficient expression of at least oneendogenous target protein wherein said cell or organism is contactedwith at least one interferor molecule capable of inhibiting the functionof at least one endogenous target protein or with a vector encoding atleast interferor molecule capable of interfering with the functionand/or presence of at least one endogenous protein. It should be notedthat the present invention also allows a target-specific knockout ofseveral different endogenous proteins due to the specificity of theinterferor molecule.

Using the protein based knockout technologies described herein, theexpression of an endogenous target protein may be inhibited in a targetcell or a target organism. The endogenous protein may be complemented byan exogenous target nucleic acid coding for the target protein or avariant or mutated form of the target protein, e.g. a gene or a cDNA,which may optionally be fused to a further nucleic acid sequenceencoding a detectable peptide or polypeptide, e.g. an affinity tag,particularly a multiple affinity tag. Variants or mutated forms of thetarget protein differ from the endogenous target protein in that theydiffer from the endogenous protein by amino acid substitutions,insertions and/or deletions of single or multiple amino acids. Thevariants or mutated forms may have the same biological activity as theendogenous target protein. On the other hand, the variant or mutatedtarget protein may also have a biological activity, which differs fromthe biological activity of the endogenous target protein, e.g. apartially deleted activity, a completely deleted activity, an enhancedactivity etc. The complementation may be accomplished by co-expressingthe polypeptide encoded by the exogenous nucleic acid, e.g. a fusionprotein comprising the target protein and the affinity tag and theinterferor molecule for knocking out the endogenous protein in thetarget cell. This co-expression may be accomplished by using a suitableexpression vector expressing both the polypeptide encoded by theexogenous nucleic acid, e.g. the tag-modified target protein and theinterferor molecule or alternatively by using a combination ofexpression vectors or alternatively the interferor molecule may contactthe target cell from the outside of the cell. Proteins and proteincomplexes which are synthesized de novo in the target cell will containthe exogenous protein, e.g. the modified fusion protein. In order toavoid suppression of the exogenous protein function with the interferormolecule, the exogenous protein must have sufficient amino aciddifferences in the aggregation region that is selected for the design ofthe interferor molecule. Alternatively, the endogenous target proteinmay be complemented by corresponding proteins from other species, or theendogenous target protein may be complemented by a splice form of saidtarget protein. The combination of knockout of an endogenous protein andrescue by using mutated, e.g. partially deleted exogenous target hasadvantages compared to the use of a knockout cell. Further, this methodis particularly suitable for identifying functional domains of thetarget protein.

In a further preferred embodiment a comparison, e.g. of gene expressionprofiles and/or proteomes and/or phenotypic characteristics of at leasttwo cells or organisms is carried out. These organisms are selectedfrom: (i) a control cell or control organism without target proteininhibition, (ii) a cell or organism with target protein inhibition and(iii) a cell or organism with target protein inhibition plus targetprotein complementation by an exogenous target nucleic acid encodingsaid target protein.

The methods of the invention are also suitable in a procedure foridentifying and/or characterizing pharmacological agents, e.g.identifying new pharmacological agents from a collection of testsubstances and/or characterizing mechanisms of action and/or sideeffects of known pharmacological agents. Thus, the present inventionalso relates to a system for identifying and/or characterizingpharmacological agents acting on at least one target protein comprising:(a) a eukaryotic cell or a eukaryotic non-human organism capable ofexpressing at least one endogenous target gene coding for said targetprotein, (b) at least one interferor molecule capable of inhibiting theexpression of said at least one endogenous target gene, and (c) a testsubstance or a collection of test substances wherein pharmacologicalproperties of said test substance or said collection are to beidentified and/or characterized. Further, the system as described abovepreferably comprises: (d) at least one exogenous target nucleic acidcoding for the target protein or a variant or mutated form or spliceform of the target protein wherein said exogenous target protein differsfrom the endogenous target protein on the amino acid level of theaggregation regions such that the function of the exogenous targetprotein is substantially less inhibited by the interferor molecule thanthe expression of the endogenous protein.

In addition, the invention also comprises cells and organisms comprisingan interferor molecule. An organism can for example be a transgenicplant which carries the genetic information that encodes an interferor.Such a transgenic plant is in a preferred embodiment a silenced plant(id est in which a particular target protein is down-regulated becauseof the presence of a specific interferor in a sub-set of cells or organsor organelles (e.g. chloroplasts) or present in all cells and organs ofsaid plant). Cells comprising an interferor can be produced bycontacting said cells or by electroporation of said cells (e.g. plantcells, plant protoplasts or plant seeds) with a particular interferormolecule. In a particular embodiment cells comprising an interferor aregenerated through transfection (or transformation) wherein theinterferor is encoded by a recombinant expression vector such as aplasmid or a viral vector.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “a target protein” means one target proteinor more than one target protein.

As used herein, the term “about” refers to a quantity, level, value,dimension, size, or amount that varies by as much as 30%, preferably byas much as 20%, and more preferably by as much as 10% to a referencequantity, level, value, dimension, size, or amount.

“Bifunctional crosslinking reagent” means a reagent containing tworeactive groups, the reagent thereby having the ability to covalentlylink two elements such as part A and part B of the interferor molecule.The reactive groups in a crosslinking reagent typically belong to theclasses of functional groups including succinimidyl esters, maleimidesand haloacetamides such as iodoacetamides. Throughout thisspecification, unless the context requires otherwise, the words“comprise”, “comprises” and “comprising” will be understood to imply theinclusion of a stated step or element or group of steps or elements butnot the exclusion of any other step or element or group of steps orelements.

By “expression vector” or “recombinant vector” is meant any autonomousgenetic element capable of directing the synthesis of an interferormolecule encoded by the vector. Such expression vectors are known topractitioners in the art.

By “derivative” is meant an interferor molecule that has been derivedfrom the basic sequence by modification, for example by conjugation orcomplexing with other chemical moieties (e.g. pegylation) or bypost-translational modification techniques as would be understood in theart. The term “derivative” also includes within its scope alterationsthat have been made to a parent sequence including additions, ordeletions that provide for functionally equivalent molecules.

By “effective amount”, in the context of modulating an activity or oftreating or preventing a condition is meant the administration of thatamount of an interferor molecule to an individual in need of suchmodulation, treatment or prophylaxis, either in a single dose or as partof a series, that is effective for modulation of that effect or fortreatment or prophylaxis of that condition. The effective amount willvary depending upon the health and physical condition of the individualto be treated, the taxonomic group of individual to be treated, theformulation of the composition, the assessment of the medical situation,and other relevant factors. It is expected that the amount will fall ina relatively broad range that can be determined through routine trials.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated polypeptide”, as used herein, refers to apolypeptide, which has been purified from the sequences which flank itin a naturally-occurring state, e.g., a self-association sequence whichhas been removed from the sequences that are normally adjacent to saidsequence. A self-association sequence (optionally coupled to a moietythat prevents aggregation) can be generated by amino acid chemicalsynthesis or can be generated by recombinant production.

The term “oligonucleotide” as used herein refers to a polymer composedof a multiplicity of nucleotide units (deoxyribonucleotides orribonucleotides, or related structural variants or synthetic analoguesthereof) linked via phosphodiester bonds (or related structural variantsor synthetic analogues thereof). An oligonucleotide is typically rathershort in length, generally from about 10 to 30 nucleotides, but the termcan refer to molecules of any length, although the term “polynucleotide”or “nucleic acid” is typically used for large oligonucleotides. The term“polynucleotide” or “nucleic acid” as used herein designates mRNA, RNA,cRNA, cDNA or DNA. The term typically refers to oligonucleotides greaterthan 30 nucleotides in length.

The term “recombinant polynucleotide” as used herein refers to apolynucleotide formed in vitro by the manipulation of nucleic acid intoa form not normally found in nature. For example, the recombinantpolynucleotide may be in the form of an expression vector. Generally,such expression vectors include transcriptional and translationalregulatory nucleic acid operably linked to the nucleotide sequence.

By “agronomically acceptable carrier” is meant a solid or liquid filler,diluent or encapsulating substance that can be safely used in topical orsystemic administration of an interferor molecule to a plant, plantseed, plant cell or plant protoplast.

“Polypeptide”, “peptide” and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues and to variants andsynthetic analogues of the same. Thus, these terms apply to amino acidpolymers in which one or more amino acid residues is a syntheticnon-naturally occurring amino acid, such as a chemical analogue of acorresponding naturally occurring amino acid, as well as tonaturally-occurring amino acid polymers.

By “recombinant polypeptide” is meant a polypeptide made usingrecombinant techniques, i.e., through the expression of a recombinant orsynthetic polynucleotide. When the chimeric polypeptide or biologicallyactive portion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the protein preparation.

The term “sequence identity” as used herein refers to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. For the purposes of the present invention, “sequence identity”will be understood to mean the “match percentage” calculated by theDNASIS computer program (Version 2.5 for windows; available from HitachiSoftware engineering Co., Ltd., South San Francisco, Calif., USA) usingstandard defaults as used in the reference manual accompanying thesoftware. “Similarity” refers to the percentage number of amino acidsthat are identical or constitute conservative substitutions. Similaritymay be determined using sequence comparison programs such as GAP(Deveraux et al. 1984, Nucleic Acids Research 12, 387-395). In this way,sequences of a similar or substantially different length to those citedherein might be compared by insertion of gaps into the alignment, suchgaps being determined, for example, by the comparison algorithm used byGAP.

The term “transformation” means alteration of the genotype of anorganism, for example a bacterium, yeast or plant, by the introductionof a foreign or endogenous nucleic acid. Vectors for transformationinclude plasmids, retroviruses and other animal viruses, YACs (yeastartificial chromosome), BACs (bacterial artificial chromosome) and thelike. By “vector” is meant a polynucleotide molecule, preferably a DNAmolecule derived, for example, from a plasmid, bacteriophage, yeast orvirus, into which a polynucleotide can be inserted or cloned. A vectorpreferably contains one or more unique restriction sites and can becapable of autonomous replication in a defined host cell including atarget cell or tissue or a progenitor cell or tissue thereof, or beintegrable with the genome of the defined host such that the clonedsequence is reproducible. Accordingly, the vector can be an autonomouslyreplicating vector, i.e., a vector that exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a linear or closed circular plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector can contain any means for assuring self-replication.Alternatively, the vector can be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. A vector system cancomprise a single vector or plasmid, two or more vectors or plasmids,which together contain the total DNA to be introduced into the genome ofthe host cell, or a transposon. The choice of the vector will typicallydepend on the compatibility of the vector with the host cell into whichthe vector is to be introduced. In a preferred embodiment, the vector ispreferably a viral or viral-derived vector, which is operably functionalin animal and preferably mammalian cells. The vector can also include aselection marker such as an antibiotic resistance gene that can be usedfor selection of suitable transformants. Examples of such resistancegenes are known to those of skill in the art and include the nptII genethat confers resistance to the antibiotics kanamycin and G418(Geneticin®) and the hph gene which confers resistance to the antibiotichygromycin B.

In the present invention a “plant expressible promoter” comprisesregulatory elements, which mediate the expression of a coding sequencesegment in plant cells. For expression in plants, the nucleic acidmolecule must be linked operably to or comprise a suitable promoterwhich expresses the gene at the right point in time and with therequired spatial expression pattern. For the identification offunctionally equivalent promoters, the promoter strength and/orexpression pattern of a candidate promoter may be analysed for exampleby operably linking the promoter to a reporter gene and assaying theexpression level and pattern of the reporter gene in various tissues ofthe plant. Suitable well-known reporter genes include for examplebeta-glucuronidase or beta-galactosidase. The promoter activity isassayed by measuring the enzymatic activity of the beta-glucuronidase orbeta-galactosidase. The promoter strength and/or expression pattern maythen be compared to that of a reference promoter (such as the one usedin the methods of the present invention). Alternatively, promoterstrength may be assayed by quantifying mRNA levels or by comparing mRNAlevels of the nucleic acid used in the methods of the present invention,with mRNA levels of housekeeping genes such as 18S rRNA, using methodsknown in the art, such as Northern blotting with densitometric analysisof autoradiograms, quantitative real-time PCR or RT-PCR (Heid et al.,1996 Genome Methods 6: 986-994). Generally by “weak promoter” isintended a promoter that drives expression of a coding sequence at a lowlevel. By “low level” is intended at levels of about 1/10,000transcripts to about 1/100,000 transcripts, to about 1/500,0000transcripts per cell. Conversely, a “strong promoter” drives expressionof a coding sequence at high level, or at about 1/10 transcripts toabout 1/100 transcripts to about 1/1000 transcripts per cell. Generally,by “medium strength promoter” is intended a promoter that drivesexpression of a coding sequence at a lower level than a strong promoter,in particular at a level that is in all instances below that obtainedwhen under the control of a 35S CaMV promoter.

The term “operably linked” as used herein refers to a functional linkagebetween the promoter sequence and the gene of interest, such that thepromoter sequence is able to initiate transcription of the gene ofinterest.

A “constitutive promoter” refers to a promoter that is transcriptionallyactive during most, but not necessarily all, phases of growth anddevelopment and under most environmental conditions, in at least onecell, tissue or organ. An “ubiquitous” promoter is active insubstantially all tissues or cells of an organism. Adevelopmentally-regulated promoter is active during certaindevelopmental stages or in parts of the plant that undergo developmentalchanges. An inducible promoter has induced or increased transcriptioninitiation in response to a chemical (for a review see Gatz 1997, Annu.Rev. Plant Physiol. Plant Mol. Biol., 48:89-108), environmental orphysical stimulus, or may be “stress-inducible”, i.e. activated when aplant is exposed to various stress conditions, or a “pathogen-inducible”i.e. activated when a plant is exposed to exposure to various pathogens.An organ-specific or tissue-specific promoter is one that is capable ofpreferentially initiating transcription in certain organs or tissues,such as the leaves, roots, seed tissue etc. For example, a“root-specific promoter” is a promoter that is transcriptionally activepredominantly in plant roots, substantially to the exclusion of anyother parts of a plant, whilst still allowing for any leaky expressionin these other plant parts. Promoters able to initiate transcription incertain cells only are referred to herein as “cell-specific”. Aseed-specific promoter is transcriptionally active predominantly in seedtissue, but not necessarily exclusively in seed tissue (in cases ofleaky expression). The seed-specific promoter may be active during seeddevelopment and/or during germination. The seed specific promoter may beendosperm/aleurone/embryo specific. Examples of seed-specific promotersare given in Qing Qu and Takaiwa (Plant Biotechnol. J. 2, 1 13-125,2004), which disclosure is incorporated by reference herein as if fullyset forth. A green tissue-specific promoter as defined herein is apromoter that is transcriptionally active predominantly in green tissue,substantially to the exclusion of any other parts of a plant, whilststill allowing for any leaky expression in these other plant parts.

The term “terminator” encompasses a control sequence which is a DNAsequence at the end of a transcriptional unit which signals 3′processing and polyadenylation of a primary transcript and terminationof transcription. The terminator can be derived from the natural gene,from a variety of other plant genes, or from T-DNA. The terminator to beadded may be derived from, for example, the nopaline synthase oroctopine synthase genes, or alternatively from another plant gene, orless preferably from any other eukaryotic gene.

“Selectable marker”, “selectable marker gene” or “reporter gene”includes any gene that confers a phenotype on a cell in which it isexpressed to facilitate the identification and/or selection of cellsthat are transfected or transformed with a nucleic acid construct of theinvention. These marker genes enable the identification of a successfultransfer of the nucleic acid molecules via a series of differentprinciples. Suitable markers may be selected from markers that conferantibiotic or herbicide resistance, that introduce a new metabolic traitor that allow visual selection. Examples of selectable marker genesinclude genes conferring resistance to antibiotics (such as nptII thatphosphorylates neomycin and kanamycin, or hpt, phosphorylatinghygromycin, or genes conferring resistance to, for example, bleomycin,streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin,geneticin (G418), spectinomycin or blasticidin), to herbicides (forexample bar which provides resistance to Basta®; aroA or gox providingresistance against glyphosate, or the genes conferring resistance to,for example, imidazolinone, phosphinothricin or sulfonylurea), or genesthat provide a metabolic trait (such as manA that allows plants to usemannose as sole carbon source or xylose isomerase for the utilisation ofxylose, or antinutritive markers such as the resistance to2-deoxyglucose). Expression of visual marker genes results in theformation of colour (for example β-glucuronidase, GUS or β-galactosidasewith its coloured substrates, for example X-Gal), luminescence (such asthe luciferin/luceferase system) or fluorescence (Green FluorescentProtein, GFP, and derivatives thereof). This list represents only asmall number of possible markers. The skilled worker is familiar withsuch markers. Different markers are preferred, depending on the organismand the selection method.

It is known that upon stable or transient integration of nucleic acidsinto plant cells, only a minority of the cells takes up the foreign DNAand, if desired, integrates it into its genome, depending on theexpression vector used and the transfection technique used. To identifyand select these integrants, a gene coding for a selectable marker (suchas the ones described above) is usually introduced into the host cellstogether with the gene of interest. These markers can for example beused in mutants in which these genes are not functional by, for example,deletion by conventional methods. Furthermore, nucleic acid moleculesencoding a selectable marker can be introduced into a host cell on thesame vector that comprises the sequence encoding the polypeptides of theinvention or used in the methods of the invention, or else in a separatevector. Cells which have been stably transfected with the introducednucleic acid can be identified for example by selection (for example,cells which have integrated the selectable marker survive whereas theother cells die).

Since the marker genes, particularly genes for resistance to antibioticsand herbicides, are no longer required or are undesired in thetransgenic host cell once the nucleic acids have been introducedsuccessfully, the process according to the invention for introducing thenucleic acids advantageously employs techniques which enable the removalor excision of these marker genes. One such a method is what is known asco-transformation. The co-transformation method employs two vectorssimultaneously for the transformation, one vector bearing the nucleicacid according to the invention and a second bearing the marker gene(s).A large proportion of transformants receives or, in the case of plants,comprises (up to 40% or more of the transformants), both vectors. Incase of transformation with Agrobacteria, the transformants usuallyreceive only a part of the vector, i.e. the sequence flanked by theT-DNA, which usually represents the expression cassette. The markergenes can subsequently be removed from the transformed plant byperforming crosses. In another method, marker genes integrated into atransposon are used for the transformation together with desired nucleicacid (known as the Ac/Ds technology). The transformants can be crossedwith a transposase source or the transformants are transformed with anucleic acid construct conferring expression of a transposase,transiently or stable. In some cases (approx. 10%), the transposon jumpsout of the genome of the host cell once transformation has taken placesuccessfully and is lost. In a further number of cases, the transposonjumps to a different location. In these cases the marker gene must beeliminated by performing crosses. In microbiology, techniques weredeveloped which make possible, or facilitate, the detection of suchevents. A further advantageous method relies on what is known asrecombination systems; whose advantage is that elimination by crossingcan be dispensed with. The best-known system of this type is what isknown as the Cre/lox system. Cre1 is a recombinase that removes thesequences located between the loxP sequences. If the marker gene isintegrated between the loxP sequences, it is removed once transformationhas taken place successfully, by expression of the recombinase. Furtherrecombination systems are the HIN/HIX, FLP/FRT and REP/STB system(Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267; Velmurugan etal., J. Cell Biol., 149, 2000: 553-566). A site-specific integrationinto the plant genome of the nucleic acid sequences according to theinvention is possible.

For the purposes of the invention, “transgenic”, “transgene” or“recombinant” means with regard to, for example, a nucleic acidsequence, an expression cassette, gene construct or a vector comprisingthe nucleic acid sequence or an organism transformed with the nucleicacid sequences, expression cassettes or vectors according to theinvention.

A transgenic plant for the purposes of the invention is thus understoodas meaning, as above, that the nucleic acids used in the method of theinvention (e.g. the artificial genes) are not present in, or originatingfrom, the genome of said plant, or are present in the genome of saidplant but not at their natural locus in the genome of said plant, itbeing possible for the nucleic acids to be expressed homologously orheterologously. However, as mentioned, transgenic also means that, whilethe nucleic acids according to the invention or used in the inventivemethod are at their natural position in the genome of a plant, thesequence has been modified with regard to the natural sequence, and/orthat the regulatory sequences of the natural sequences have beenmodified. Transgenic is preferably understood as meaning the expressionof the nucleic acids according to the invention at an unnatural locus inthe genome, i.e. homologous or, heterologous expression of the nucleicacids takes place. Preferred transgenic plants are mentioned herein.

The term “expression” or “gene expression” means the transcription of aspecific gene or specific genes or specific genetic construct. The term“expression” or “gene expression” in particular means the transcriptionof a gene or genes or genetic construct into structural RNA (rRNA, tRNA)or mRNA with or without subsequent translation of the latter into aprotein. The process includes transcription of DNA and processing of theresulting mRNA product.

The term “increased expression” or “overexpression” as used herein meansany form of expression that is additional to the original wild-typeexpression level. For the purposes of this invention, the originalwild-type expression level might also be zero, i.e. absence ofexpression or immeasurable expression.

Methods for increasing expression of genes or gene products are welldocumented in the art and include, for example, overexpression driven byappropriate promoters (as described herein before), the use oftranscription enhancers or translation enhancers. Isolated nucleic acidswhich serve as promoter or enhancer elements may be introduced in anappropriate position (typically upstream) of a non-heterologous form ofa polynucleotide so as to upregulate expression of a nucleic acidencoding the polypeptide of interest. If polypeptide expression isdesired, it is generally desirable to include a polyadenylation regionat the 3′-end of a polynucleotide coding region. The polyadenylationregion can be derived from the natural gene, from a variety of otherplant genes, or from T-DNA. The 3′ end sequence to be added may bederived from, for example, the nopaline synthase or octopine synthasegenes, or alternatively from another plant gene, or less preferably fromany other eukaryotic gene.

An intron sequence may also be added to the 5′ untranslated region (UTR)or the coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold (Buchman and Berg(1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200). Such intron enhancement of gene expression is typicallygreatest when placed near the 5′ end of the transcription unit. Use ofthe maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron areknown in the art. For general information see: The Maize Handbook,Chapter 1 16, Freeling and Walbot, Eds., Springer, N.Y. (1994).

The term “introduction” or “transformation” as referred to hereinencompass the transfer of an exogenous polynucleotide into a host cell,irrespective of the method used for transfer. Plant tissue capable ofsubsequent clonal propagation, whether by organogenesis orembryogenesis, may be transformed with a genetic construct of thepresent invention and a whole plant regenerated there from. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristem, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand hypocotyl meristem). The polynucleotide may be transiently or stablyintroduced into a host cell and may be maintained non-integrated, forexample, as a plasmid. Alternatively, it may be integrated into the hostgenome. The resulting transformed plant cell may then be used toregenerate a transformed plant in a manner known to persons skilled inthe art.

The transfer of foreign genes into the genome of a plant is calledtransformation. Transformation of plant species is now a fairly routinetechnique. Advantageously, any of several transformation methods may beused to introduce the gene of interest into a suitable ancestor cell.The methods described for the transformation and regeneration of plantsfrom plant tissues or plant cells may be utilized for transient or forstable transformation. Transformation methods include the use ofliposomes, electroporation, chemicals that increase free DNA uptake,injection of the DNA directly into the plant, particle gun bombardment,transformation using viruses or pollen and microprojection. Methods maybe selected from the calcium/polyethylene glycol method for protoplasts(Krens, F. A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987)Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R.D. et al. (1985) Bio/Technol 3, 1099-1 102); microinjection into plantmaterial (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA orRNA-coated particle bombardment (Klein T M et al., (1987) Nature 327:70) infection with (non-integrative) viruses and the like. Transgenicplants, including transgenic crop plants, are preferably produced viaAgrobacterium-mediated transformation. An advantageous transformationmethod is the transformation in planta. To this end, it is possible, forexample, to allow the agrobacteria to act on plant seeds or to inoculatethe plant meristem with agrobacteria. It has proved particularlyexpedient in accordance with the invention to allow a suspension oftransformed agrobacteria to act on the intact plant or at least on theflower primordia. The plant is subsequently grown on until the seeds ofthe treated plant are obtained (Clough and Bent, Plant J. (1998) 16,735-743). Methods for Agrobacterium-mediated transformation of riceinclude well known methods for rice transformation, such as thosedescribed in any of the following: European patent applicationEP1198985, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al.(Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2):271-282, 1994), which disclosures are incorporated by reference hereinas if fully set forth. In the case of corn transformation, the preferredmethod is as described in either Ishida et al. (Nat. Biotechnol 14(6):745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), whichdisclosures are incorporated by reference herein as if fully set forth.Said methods are further described by way of example in B. Jenes et al.,Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineeringand Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993)128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42(1991) 205-225). The nucleic acids or the construct to be expressed ispreferably cloned into a vector, which is suitable for transformingAgrobacterium tumefaciens, for example pBin19 (Bevan et al (1984) Nucl.Acids Res. 12-8711). Agrobacteria transformed by such a vector can thenbe used in known manner for the transformation of plants, such as plantsused as a model, like Arabidopsis or crop plants such as, by way ofexample, tobacco plants, for example by immersing bruised leaves orchopped leaves in an agrobacterial solution and then culturing them insuitable media. The transformation of plants by means of Agrobacteriumtumefaciens is described, for example, by Hofgen and Willmitzer in Nucl.Acid Res. (1988) 16, 9877 or is known inter alia from F. F. White,Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol.1, Engineering and Utilization, eds. S. D. Kung and R. Wu, AcademicPress, 1993, pp. 15-38.

In addition to the transformation of somatic cells, which then have tobe regenerated into intact plants, it is also possible to transform thecells of plant meristems and in particular those cells which developinto gametes. In this case, the transformed gametes follow the naturalplant development, giving rise to transgenic plants. Thus, for example,seeds of Arabidopsis are treated with agrobacteria and seeds areobtained from the developing plants of which a certain proportion istransformed and thus transgenic [Feldman, K A and Marks M D (1987). MolGen Genet 208:1-9; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell,eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp.274-289]. Alternative methods are based on the repeated removal of theinflorescences and incubation of the excision site in the center of therosette with transformed agrobacteria, whereby transformed seeds canlikewise be obtained at a later point in time (Chang (1994). Plant J. 5:551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, anespecially effective method is the vacuum infiltration method with itsmodifications such as the “floral dip” method. In the case of vacuuminfiltration of Arabidopsis, intact plants under reduced pressure aretreated with an agrobacterial suspension [Bechthold, N (1993). CR AcadSci Paris Life Sci, 316: 1 194-1 199], while in the case of the “floraldip” method the developing floral tissue is incubated briefly with asurfactant-treated agrobacterial suspension [Clough, S J and Bent A F(1998) The Plant J. 16, 735-743]. A certain proportion of transgenicseeds are harvested in both cases, and these seeds can be distinguishedfrom non-transgenic seeds by growing under the above-described selectiveconditions. In addition the stable transformation of plastids is ofadvantages because plastids are inherited maternally is most cropsreducing or eliminating the risk of transgene flow through pollen. Thetransformation of the chloroplast genome is generally achieved by aprocess which has been schematically displayed in Klaus et al., 2004[Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to betransformed are cloned together with a selectable marker gene betweenflanking sequences homologous to the chloroplast genome. Thesehomologous flanking sequences direct site specific integration into theplastome. Plastidal transformation has been described for many differentplant species and an overview is given in Bock (2001) Transgenicplastids in basic research and plant biotechnology. J Mol Biol. 2001Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towardscommercialization of plastid transformation technology. TrendsBiotechnol. 21, 20-28. Further biotechnological progress has recentlybeen reported in form of marker free plastid transformants, which can beproduced by a transient co-integrated maker gene (Klaus et al., 2004,Nature Biotechnology 22(2), 225-229).

The genetically modified plant cells can be regenerated via all methodswith which the skilled worker is familiar. Suitable methods can be foundin the abovementioned publications by S. D. Kung and R. Wu, Potrykus orHofgen and Willmitzer.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant. To select transformed plants, the plant material obtained in thetransformation is, as a rule, subjected to selective conditions so thattransformed plants can be distinguished from untransformed plants. Forexample, the seeds obtained in the above-described manner can be plantedand, after an initial growing period, subjected to a suitable selectionby spraying. A further possibility consists in growing the seeds, ifappropriate after sterilization, on agar plates using a suitableselection agent so that only the transformed seeds can grow into plants.Alternatively, the transformed plants are screened for the presence of aselectable marker such as the ones described above.

Following DNA transfer and regeneration, putatively transformed plantsmay also be evaluated, for instance using Southern analysis, for thepresence of the gene of interest, copy number and/or genomicorganisation. Alternatively or additionally, expression levels of thenewly introduced DNA may be monitored using Northern and/or Westernanalysis, both techniques being well known to persons having ordinaryskill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedand homozygous second-generation (or T2) transformants selected, and theT2 plants may then further be propagated through classical breedingtechniques. The generated transformed organisms may take a variety offorms. For example, they may be chimeras of transformed cells andnon-transformed cells; clonal transformants (e.g., all cells transformedto contain the expression cassette); grafts of transformed anduntransformed tissues (e.g., in plants, a transformed rootstock graftedto an untransformed scion).

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,leaves, roots (including tubers), flowers, and tissues and organs,wherein each of the aforementioned comprise the gene/nucleic acid ofinterest. The term “plant” also encompasses plant cells, suspensioncultures, callus tissue, embryos, meristematic regions, gametophytes,sporophytes, pollen and microspores, again wherein each of theaforementioned comprises the gene/nucleic acid of interest.

Plants that are particularly useful in the methods of the inventioninclude in particular monocotyledonous and dicotyledonous plantsincluding fodder or forage legumes, ornamental plants, food crops, treesor shrubs selected from the list comprising Acer spp., Actinidia spp.,Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera,Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annonaspp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagusofficinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avenabyzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola,Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris,Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseedrape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica,Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissamacrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceibapentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrusspp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorussp, Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus,Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodiumspp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloaspp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusinecoracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptussp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea,Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycinespp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum,Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscusspp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglansspp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linumusitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinusspp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum,Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp.,Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica,Manihot spp., Manilkara zapota, Medicago sativa, Malilotus spp., Menthaspp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp.,Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp.(e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicumvirgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Perseaspp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleumpratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp.,Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunusspp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp.,Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubusspp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamumspp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanumintegrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp.,Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao,Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticumspp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum,Triticum hybemum, Triticum macha, Triticum sativum, Triticum monococcumor Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vacciniumspp., Vida spp., Vigna spp., Viola odorata, Vitis spp., Zea mays,Zizania palustris, Ziziphus spp., amongst others.

The choice of suitable control plants is a routine part of anexperimental setup and may include corresponding wild type plants orcorresponding plants without the gene of interest. The control plant istypically of the same plant species or even of the same variety as theplant to be assessed. The control plant may also be a nullizygote of theplant to be assessed. Nullizygotes are individuals missing the transgeneby segregation. A “control plant” as used herein refers not only towhole plants, but also to plant parts, including seeds and seed parts.

The term “expression cassette” refers to any recombinant expressionsystem for the purpose of expressing a nucleic acid sequence of theinvention in vitro or in vivo, constitutively or inducibly, in any cell,including, in addition to plant cells, prokaryotic, yeast, fungal,insect or mammalian cells. The term includes linear and circularexpression systems. The term includes all vectors. The cassettes canremain episomal or integrate into the host cell genome. The expressioncassettes can have the ability to self-replicate or not (i.e., driveonly transient expression in a cell). The term includes recombinantexpression cassettes that contain only the minimum elements needed fortranscription of the recombinant nucleic acid.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, useful methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features andadvantages of the invention will be apparent from the detaileddescription and from the claims.

EXAMPLES Introduction

In order to test the protein interference technology in planta, acytosolic player of the brassinosteroids (BR) signalling pathway hasbeen selected. BR are steroidal hormones affecting many cellularprocesses involved in organ growth and plant development includingvascular differentiation, senescence, male fertility, flowering,photomorphogenesis, tolerance to biotic and abiotic stresses (Bajguz andHayat (2009) Plant Physiol. Biochem 47(1): 1-8). They are also acting onagronomically interesting traits in crop plants as tiller number, leafsize, and leaf angle (Morinake Y et al (2006) Plant Physiol 141(3):924-31. As an example, the tissue-specific expression of the sterol C-22hydroxylases, an enzyme controlling BR hormone levels, can enhance thegrain filling in rice (Wu C Y et al (2008) Plant Cell 20(8): 2130-45).

In Arabidopsis BR are perceived by the plasma membrane leucine-richrepeat (LRR) receptor-like kinases (RLK) BRASSINOSTEROID INSENSITIVE 1(BRI1) which gets activated upon BR binding and associates withBRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) inducing sequentialtransphosphorylation events by which the fully activated BRI1 canfurther phosphorylate BR-SIGNALING KINASES (BSKs). Then thephosphorylated BSKs are released from the receptor complex and bind tothe BRI1 SUPPRESSOR 1 (BSU1) phopsphates, presumably enhancing itsactivity. Activated BSU1 inhibits Brassinosteroid Insensitive 2 (BIN2)and other kinases belonging to the glycogen synthase kinase-3 (GSK3)family by dephosphorylating its phospho-tyrosine residue.Unphopsphorylated BIN2 allows accumulation of active unphosphorylatedBRASSINAZOLE RESISTANT1 (BZR1) and BZR2/bri1-EMS-SUPPRESSOR1 (BZR2/BES1)transcription factors in the nucleus. Active BZR1 and BZR2/BES1 bind togenomic DNA to regulate BR-target gene expression, thereby modulatinggrowth and development of plants.

In this pathway a good putative target has been identified in the BRnegative regulator BIN2 as we expected that interfering with it by usingthe protein interference technology would result in agronomicalllyinteresting phenotypic changes. To this end the protein aggregationknock out efficiency was correlated with the phenotypic alterationsobserved by scoring growth parameters as leaf shape and plant height.These parameters have important agronomic effects on crop yield as ithas been shown in rice knock-out brassinosteroid signalling mutantswhich have higher yields in dense planting conditions.

In the present study two main biological questions are addressed; atfirst whether it is possible to visualize and evaluate the β-aggregationphenomenom in plants, and then if the targeting of a specific protein ofinterest by aggregating baits is achievable by proving its functionalknock-out.

1. Design of BIN2 Interferor Expressing Constructs

The GSK3-like kinase BIN2 was selected as a suitable plant targetbecause of its cytosolic and nuclear localization that could permit atargeting by the expressed aggregating peptides (i.e. interferorpeptides). We identified two short amino acidic stretches in the BIN2primary amino acid sequence (see FIG. 1 a) with the TANGO algorithm.These two beta-aggregation prone amino acid sequences were predicted tohave a propensity to aggregate higher than 50%. These interferorpeptides, also designated herein further as baits, cover the BIN2regions from 44-55aa (bait44) and in the kinase domain from 249-257aa(bait249). For experimental reasons we decided to focus our study onbait249. To induce BIN2 aggregation, several constructs wherein bait249was C-terminally fused to eGFP fluorescent protein were engineered inplant binary expression vectors. These bait249 expressing constructshave been engineered with and without positively charged amino acids(herein designated as gatekeepers); the construct comprising gatekeeperswas designated as “bait249R” and the construct without these gatekeeperswas designated as “bait249”. The latter constructs were made to evaluatetheir role in enhancing the cytosolic localization of the expressedbait. In addition, a synthetic sequence know to boost the aggregationprocess (herein designated as booster) has been inserted between thebait and the eGFP through a linker sequence (the specific sequences aredepicted in FIG. 1 b).

To evaluate which biochemical features of the aggregating peptides weremore optimal in achieving aggregates formation and specific targeting,different variants of the bait249 have also been further engineered. Twoconstructs expressing the bait249, flanked by 5-7aa naturally flankingthe bait sequence in the BIN2 protein, inserted either in single copy(designated as bait249NF) or in tandem repeat (designated asbait249NF_Tand) have been generated. In this second set of vectors nobooster of aggregation was inserted (see FIG. 1 c).

2. Visualization of Bait249 Aggregation by Transient Expression in N.benthamiana Leaves

The ability of the different baits to induce the formation of aggregatesin plants has been initially checked with the Confocal Laser ScanningMicroscope (CLSM) in a transient expression system by overexpressing thebaits through Agrobacterium tumefaciens-mediated infiltration inNicotiana benthamiana leaves. It was observed that the absence ofgatekeepers in the bait249 vector strongly induced the formation ofinsoluble inclusion bodies rather than cytosolic expression (aswitnessed in FIG. 2 a). Conversely the bait249R induced a very strongcytosolic perinuclear aggregation as clearly shown by comparing theeffect with the free GFP localization pattern (see FIG. 2 b,e). Thesignal detected was more uniform than for bait249 and no inclusionbodies were identified. On the other hand the second set of constructsused in this study showed a clear presence of perinuclear aggregates inboth versions, wherein the construct expressing the bait in tandemrepeats (bait249NF_Tand) was the strongest aggregating construct asobserved with the CLSM (see FIG. 2 c,d). In these constructs it appearsthat the extra flanking amino acids, added to the bait sequences, playan important role in achieving cytosolic aggregation.

3. Bait259 Co-Localizes and Physically Interacts with the BIN2 TargetProtein in N. benthamiana Cells

A transient co-localization assay in N. benthamiana leaves of BIN2 andbait249 has been performed to have a fast indication of the baittargeting and co-aggregation tendency towards its target. Thereto,leaves were co-injected with A. tumefaciens strains expressing thebait249 strongest aggregating version as observed at the CLSM level,i.e. bait249NF_Tand fused to a RFP fluorescent protein and BIN2 proteinfused to eGFP. The expression of the bait249NF_Tand was induced 24 hoursbefore the subsequent fluorescence microscopy evaluation. CLSM analysisof leaves 4-5 days after injection showed a clear co-localizationbetween the bait and the target evidenced both by overlapping expressionpatterns than by the calculated co-localization Mander's coefficientswith values higher than 0.8. The formation of co-localizing cytosolicaggregates was also assessed in fluorescence microscopy (see FIG. 3). Ina next step, after CLSM confirmation of co-localization and proteinaggregation between the bait-GFP proteins and the BIN2 target, theirstable physical interaction in a transient expression system was alsoassessed by co-immunoprecipitation (co-IP) experiments. To circumventthe lack of a specific BIN2 antibody the target protein was N-terminallyfused to a haemaglutinin (HA) immunological tag. N. benthamiana leaveswere then co-injected with Agrobacterium strains transformed withdifferent bait249-GFP expressing vectors and the BIN2-HA was expressedunder control of the 35S promoter. Both the bait249 with and withoutgatekeepers (and with booster of aggregation) and the bait249 in singleand in tandem repeats (without booster) were tested. The co-IPexperiment was performed by pull down of the GFP tagged baits byanti-GFP agarose coupled beads and subsequent Western blot detection wasachieved with an anti-HA monoclonal antibody. Different negativecontrols were used: i) to test for unspecific binding of GFP to thebeads a freeGFP encoding vector was co-injected with BIN2HA; ii) to testfor unspecific binding of the synthetic booster to the beads a vectorencoding only the booster and the linker sequence fused to GFP has beenengineered and co-injected with BIN2-HA, iii) to test for unspecificbinding either of the BIN2 protein or of the HA tag to the beads theBIN2-HA construct was injected alone; and iv) also a wild type plantextract has been used as additional negative control. The co-IPexperiment indicated a positive interaction for any version of thebait249 tested with BIN2 thereby strongly demonstrating that the baitand the target can interact via the formation of a specific biochemicalinteraction, i.e. a cross-β-sheet-mediated aggregation (see FIG. 4).This result confirmed what was observed in the co-localization assaysthereby generating proof of concept that a physical interaction betweenthe two partners occurs in an in vivo system.

4. Assessing the Efficiency of Protein Aggregation in ArabidopsisTransgenic Plants

After transformation of the several GFP tagged bait249 expressingconstructs the efficiency of aggregation was also monitored intransgenic Arabidopsis plants.

The evaluation of the induced aggregator complexes was assessed byimaging the GFP fluorescent protein at the CLSM microscope in eachhomozygous line.

It was observed that the 35S::bait249R-GFP and the35S::bait249NF_Tand-GFP expressing lines showed the strongestsubcellular GFP expression pattern with a clear perinuclear aggregationin different seedlings tissues (cotyledons, petioles, hypocotyls, androot) (see FIG. 5 a-d, e-h).

In contrast, for the Arabidopsis plants comprising the 35S::bait249-GFPconstruct, the absence of gatekeepers impaired the expression of anycytosolically localized aggregates in Arabidopsis cells leading to aweaker expression of the reporter protein and the formation ofround-shaped insoluble bodies in the cells, as was also observed intransiently transformed leaves of Nicotiana. The 35S::bait249NF-GFPexpression pattern was weaker than for the bait in tandem (see FIG. 6a-h). For the abovementioned reasons the constructs 35S::bait249-GFP and35S::bait249NF-GFP have not been considered for further functionalanalyses.

To investigate at the subcellular level the 35S::bait249R-GFP and35S::bait249NF_Tand-GFP localization pattern in Arabidopsis cells,Transmission Electron Microscopy (TEM) was performed on seedlings 8 daysafter sowing (D.A.S.) stably expressing these constructs. The cytosolicbait-GFP localization pattern of the lines selected for further analyseshave been confirmed by immunogold labeling experiments. In this approachlabeling of hypocotyls and root cells in 35S::bait249R-GFP line with ananti-GFP antibody resulted in specific subcellular localization of thebait mainly in the cytosol of cells belonging to the root elongationarea. The aggregating proteins appeared to be arranged both in fibrillarstructures than in clustered agglomerations indicating that theaggregates can acquire different shapes in the cells (FIG. 7 a-c). Golgistacks were free from gold particles (see FIG. 7 c) that instead appearto be more abundant in membrane-like structures (i.e. the ER) (FIG. 14b). The presence of free cytosolic bait249RGFP protein was also rarelyfound. For 35S::bait249NF_Tand-GFP a massive cytosolic and perinuclearlocalization was noticed in palisade cells in cotyledons and in rootelongation area cells and no peculiar aggregator complexes shapes weredetected (FIG. 7 d-e).

Biochemical confirmation of aggregator proteins levels has been assessedfor each Arabidopsis transformed line by Native-PAGE electrophoresis andsubsequent Western blot analysis with an anti-GFP monoclonal antibody(see FIG. 8 a).

The biochemical nature of the aggregates has been then further analyzedby Fourier Transform-Infra Red (FT-IR) Spectroscopy after theirimmunoprecipitation (IP) with anti-GFP antibody. FT-IR spectra clearlyshowed two peaks in absorbance at 1616 and 1680λ values indicating ahigh content of β-sheets aggregates in 35S::bait249R-GFP and35S::bait249-GFP lines, besides their different subcellular localizationpattern (see FIG. 8 b). For lines 35S::bait249NF-GFP and35S::bait249NF_Tand-GFP a slighter increase in 1616 and 1680 absorptionvalues was detected indicating a β-sheet content in theimmunoprecipitated material, although at a lesser extent than for thepreviously analyzed lines (FIG. 8 c).

4.1 Phenotype of Transgenic Arabidopsis Plants

The homozygotic bait249 expressing lines were then further analyzed bothin vitro and in soil for the appearance of phenotypes showing that aknock-down in BIN2 was occurring. The 35S::bait249R-GFP and35S::bait249NF_Tand-GFP transgenic seedlings, vertically grown for 8days in vitro, had longer roots and hypocotyls than the untransformedline Col-0; this observation was also confirmed by quantification withthe ImageJ software (FIG. 9 a). The statistical evaluation performedindicated a statistical significance between Col-0 and transgenic lines.One month old transgenic plants grown in soil also resulted in biggerindividuals with respect to Col-0 (FIG. 9 a). The 35S::bait249-GFP and35S::bait249NF-GFP transgenic seedlings did not show any phenotypicaldifference with Col-0 neither in vitro nor in soil conditions and werenot included in the further analysis.

In a next step, to provide further evidence that the BIN2 function isaffected by its specific aggregation, 35S::bait249R-GFP and35S::bait249NF-GFP lines were examined for resistance to thebrassinosteroid biosynthesis inhibitor, brassinazole (BRZ). As apositive control the triple mutant knock-out in BIN2 and its two closehomologues (atsk22 and atsk23) was used (Vert G and Chory J (2006)Nature 441 (7089): 96-100). We expected that if the function of BIN2 wasaffected it would result in plants being at least partially resistant tobrassinazole (please note that the triple mutant (Vert G and Chory J(2006) Nature 441 (7089): 96-100) is resistant to brassinazole. We couldindeed show that the transgenic lines showed a partial resistance to theinhibitor brassinazole, as quantified in terms of hypocotyl length (seeFIG. 9 b).

4.2 Gene Expression Changes in Transgenic Arabidopsis Plants

In a quantitative real-time PCR (qRT-PCR) analysis on BRs-related DWF4and CPD gene expression we demonstrated a decreased expression level ofDWF4 in the two aggregator lines. In the case of the CPD geneexpression, an effect was only observed for 35S:bait249NF_Tand:GFP,indicating a feedback inhibition, and thus an activated BRs signaling(see FIG. 10 a). Accordingly, the analysis of the relative expressionlevels of a BR-responsive transcription factor from the NAC familyshowed a slightly increased expression for the 35S:bait249NF_Tand:GFPconstruct (see FIG. 10 a).

Besides BR-related genes, the effect of 35S::bait249R-GFP and35S::bait249NF_Tand-GFP expression in transgenic Arabidopsis lines inthe induction of the expression of chaperone proteins was alsomonitored. Interestingly, the two aggregator lines, but in particularthe transgenic plant expressing the bait249 in tandem repeats(35S::bait249NF_Tand-GFP) showed higher (induced) expression levels ofHSP70, HSP90-1, HSP101, HSC70-1, HSC70-2 and HSC70-3 genes (see FIG. 10b).

4.3 Morphological Evaluation of Transgenic Arabidopsis Plants

In addition a morphological evaluation at the transmission electronmicroscopy (TEM) level of the transgenic lines was performed to monitora possible cytotoxic effect of the aggregator constructs at thesubcellular level. With this approach no peculiar alteration in size andshapes of cells and subcellular organelles could be observed indifferent tissues of the 35S::bait249R-GFP line (see FIG. 11).Occasionally a larger amount of plastoglobuli was found in chloroplastsof the transgenic line. The latter phenomenon is usually an indicationof stress which in the to present case could also be caused by the invitro growth conditions on nylon meshes. TEM evaluation is alsoperformed on the homozygotic line expressing the bait249 in tandemrepeats without booster of aggregation.

To assess the co-localization of the target protein and the bait instably transformed Arabidopsis lines, we aimed to visualize theco-localization between 35S::BIN2-GFP with the strongly expressedaggregator variant bait249NF_Tand fused to tagRFP fluorescent proteinexpressed under an inducible promoter (pMDC::bait249NF_Tand-RFP). Tothis end the best 35S::BIN2-GFP expressing line was super-transformedwith the estradiol inducible pMDC::bait249NF_Tand-RFP construct. Theco-localization assays were performed on the primary transformants after24 hours of bait249NF_Tand induction and then analyzed at the CLSM. Theconfocal analysis of 8 D.A.S. transformed seedlings showed a clearoverlapping localization pattern of the bait and the target (see FIG.12). The intensity of the co-localization observed was also quantifiedwith an ad hoc software (ImageJ MBF) which released Mander'scoefficients close to 1 for all the pictures processed, meaning that ahigh co-localization between the bait and the target protein occurred.

Materials and Methods for the Examples Section 1. Cloning of Bait259Aggregator in Plant Compatible Gateway Vectors

By using the TANGO prediction tool (http://tango.switchlab.org/), twoaggregator peptides that target two different bait regions (44-55aa:RVVGTGSFGIVFK (SEQ ID NO:1); 249-257aa: QLVEIIKVL (SEQ ID NO:2)) in theBIN2 protein were initially selected. For BIN2 region 249-257aa, theaggregator constructs were designed both with (bait249R: RQLVEIIKVLR(SEQ ID NO:3)) and without (bait249: QLVEIIKVL) flanking gatekeepers,represented by positively charged arginine residues.

The respective bait sequences were C-terminally fused to a syntheticsequence booster of aggregation (QWQNSTLIVLQNSTVIFEQNSTVIFEQN (SEQ IDNO:4)) by PCR analysis, introducing a flexible linker sequenceKPAGAAKPGAAG (SEQ ID NO:5).

By using the rationale of checking which bait amino-acidic modificationscould lead to better targeting of the BIN2 protein, two other vectorsexpressing the bait249 were then generated. The bait249 was modified byadding 5-7aa naturally flanking the 249-257aa region in BIN2 (bait249NF:ENAVDQLVEIIKVL GTPTREE (SEQ ID NO:6)), as well as 6 amino acids (MADDKE(SEQ ID NO:7)) corresponding to the beginning of the BIN2 proteinsequence. The flexible linker sequence has been changed toAGSPKGAPAAKGSGA (SEQ ID NO:8) and the booster sequence removed. In oneconstruct the bait has been inserted in tandem repeat (bait249NF_Tand:ENAVDQLVEIIKVL GTPTREEENAVDQLVEIIKVLGTPTREE (SEQ ID NO:9)). Theresulting DNA sequences were Gateway cloned in pDONR221 entry vectors.After sequence confirmation, the inserts were transferred to thepK7WG2,0 (Karimi et al. 2007) destination vector to generate plantbinary vectors containing the 35S promoter and the heterologous sequenceC-terminally fused to eGFP fluorescent tag. The bait249NF_Tand was alsocloned in a Gateway pMDC-m13GW vector containing an estradiol induciblepromoter (Curtis and Grossniklaus 2003) and the heterologous sequencehas been inserted C-terminally fused to tagRFP fluorescent protein(pMDC::bait249NF_Tand-tagRFP). The 35S::BIN2-HA vector has beenengineered by using pKWG2,0 destination vector to generate plant binaryvectors containing the 35S promoter and the heterologous sequence thatwas C-terminally fused by homologous recombination to an HA fluorescenttag.

2. Plant Materials and Growth Conditions

N. benthamiana plants were grown directly in soil under a 16 L/8 Dphotoperiod at 21° C. for 45 days and infiltrated before flowering.

Arabidopsis thaliana L. (Heyhn.) (Columbia ecotype, Col-0) seedlingswere stratified for 2 days at 4° C. and germinated in square plates onvertical half-strength Murashige & Skoog (MS) medium (Duchefa)containing 1% sucrose and 0.8% agar, pH 5.9, at 22° C. in a 16-h/8-hlight-dark cycle with a light intensity of 80 to 100 mE m⁻² s⁻¹ suppliedby cool-white fluorescent tubes (Spectralux Plus 36W/840; Radium) exceptwhen indicated. Seedlings grown in vitro for 21 days were transferred tosoil in growth room with similar light and temperature conditions. Thefollowing mutant line was used in this study: bin2-3/atsk22/atsk23triple mutant (Vert and Chory 2006). Previously described transgeniclines used in the study are: pBIN2::BIN2-GFP and 35S::BIN2-GFP (Vert andChory, 2006). Brassinazole (BRZ) was purchased from TCI EUROPE N.V.(Belgium), 24-Epibrassinolide (BL) from Fuji Chemical Industries(Japan). The expression of pMDC::bait249NF_Tand-RFP was induced byadding or infiltrating 20 μM Estradiol (Sigma) for 24 h.

3. Plants Transformation

To generate stable A. thaliana transgenic plants, the engineeredconstructs were transformed in A. tumefaciens C58C1 strain. Suspensionsof the transformed bacterial strains were then used to dip A. thalianaCol-0 wild-type floral buds. Primary transformants were selected bygerminating the seeds of the transformed flowers on antibiotic-selectivemedium. Trough 3:1 segregation analysis of the next generationhomozygotic transgenic lines were further isolated. Foragroinfiltrations N. benthamiana leaves were injected with A.tumefaciens strains C58C1(pCH32) transformed with vectors35S::bait249R-GFP, 35S::bait249-GFP, 35S::bait249NF-GFP,35S::bait249NF_Tand-GFP, pMDC:bait249NF_Tand-RFP together with 35S::P19,encoding the silencing inhibitor protein p19 derived from the TomatoBushy Stunt Virus (Voinnet et al. 2003).

The strains were used to co-infiltrate, with a syringe without needle,the abaxial side of N. benthamiana leaves following a previouslypublished protocol with minor modifications (English et al. 1996).Briefly, bacteria were grown overnight at 28° C. in YEB mediumcontaining 10 mM 2-(N-morpholino)ethanesulfonic acid pH 5.5 and 20 μMacetosyringone. At the optical density (O.D.) of 0.8 bacteria werepelletted, resuspended in 10 mM MES, 10 mM MgCl₂, 100 μM acetosyringoneand kept at room temperature for 3 hours before infiltration.

4. Imaging and Image Analysis

Seedlings were imaged on a laser scanning confocal microscope (OlympusFluoView 1000) with a 20× or a 60× water immersion lens, NA1.2. Imageanalysis was done with Olympus FluoView FV10-ASW software. Forco-localization experiments, Mander's overlap coefficient calculationswere done with ImageJ MBF software.

5. Tissue Fixation and Immunological Labeling for Electron Microscopy

For morphological studies, fragments (1-2 mm²) of cotyledons, hypocotylsand roots of 35S::bait249R-GFP and 35S::bait249NF_Tand-GFP 8 days aftersowing (D.A.S.) seedlings were immersed in a fixative solution of 3%paraformaldehyde and 2.5% glutaraldehyde and postfixed in 1% OsO₄ with1.5% K₃Fe(CN)₆ in 0.1 M NaCacodylate buffer, pH 7.2. Samples weredehydrated through a graded ethanol series, including a bulk stainingwith 2% uranyl acetate at the 50% ethanol step followed by embedding inSpurr's resin. Ultrathin sections were made using an ultramicrotome(Leica EM UC6) and post-stained in a Leica EM AC20 for 40 min in uranylacetate at 20° C. and for 10 min in lead stain at 20° C. Grids wereviewed with a JEM 1010 transmission electron microscope (JEOL, Tokyo,Japan) operating at 80 kV. For immunocytochemical detection, fragments(1-2 mm²) of cotyledons, hypocotyls and roots of 35S::bait249R-GFP and355::bait249NF_Tand-GFP 8 D.A.S. seedlings were immersed in a fixativesolution of 2.5% paraformaldehyde and 0.3% glutaraldehyde in 0.1 MNaCacodylate buffer, pH 7.2. Samples were dehydrated through a gradedethanol series and infiltrated stepwise over 3 d at 4° C. in LR-White,hard grade (London Resin), followed by embedding in capsules.Polymerization was done by UV illumination for 24 h at 4° C. followed by16 h at 60° C. Ultrathin sections of gold interference colour were cutwith an ultramicrotome (Leica EM UC6) and collected on formvar-coatedcopper slot grids. All steps of immunolabeling were performed in a humidchamber at room temperature. Grids were floated upside down on 25 μl ofblocking solution (5% (w/v) bovine serum albumin (BSA), for 30 minfollowed by washing five times for 5 min each times with 1% BSA in PBS.Incubation in a 1:50 dilution (1% BSA in PBS) of primary antibodiesanti-GFP rabbit (AbCam) for 60 min was followed by washing five timesfor 5 min each time with 0.1% BSA in PBS. The grids were incubated withPAG 10 nm 1:60 dilution (1% BSA in PBS) (Cell Biology, UtrechtUniversity, The Netherlands) and washed twice for 5 min each time with0.1% BSA in PBS, PBS, and double-distilled water. Sections werepost-stained in a Leica EM AC20 for 40 min in uranyl acetate at 20° C.and for 10 min in lead citrate at 20° C. Grids were viewed with a JEM1010 transmission electron microscope (Jeol Ltd., Tokyo, Japan)operating at 80 kV.

6. Electrophoresis and Western Blot

Total soluble proteins (TSP) were extracted from Arabidopsis 8 D.A.S.seedlings or fragments of N. benthamiana agroinfiltrated leaves weregrinded in a mortar and rinsed with ice-cold 0.01 M phosphate bufferedsaline (PBS; 10 mM Na₂HPO₄, 10 mM NaH₂PO₄, 150 mM NaCl, pH 7.2), addedof proteases inhibitors (Complete® EDTA free, Roche, Germany). Thehomogenates were then centrifuged at 20,000×g for 20′ at 4° C.Supernatants were quantified for protein content with the Bradford assay(Micro Assay kit, Bio-Rad Laboratories Inc., Hercules, Calif., U.S.A.).

The co-immunoprecipitation experiment has been carried out by usingagarose-coupled anti-GFP beads (Chromotek) according to the manufacturerinstructions.

Whole protein extracts or IP products were fractionated either bySDS-PAGE (Biorad) or BN-PAGE (NativePAGE system, Invitrogen) followingthe product manuals. For SDS-PAGE, approximately 60 μg of TSP weredenatured at 95° C. for 10 min in the presence of 1% SDS and 0.1M DTTand then fractionated by 10%, 12% or 15% (w/v) SDS-PAGE gels in TGSrunning buffer (25 mM Tris, 192 mM glycine and 0.1% SDS). Membranes wereblocked overnight with 5% (w/v) nonfat milk in PBS at 4° C. beforeimmunoblotting.

For BN-PAGE, protein extract was added with 20% glycerol and 5 mMCoomassie G-250 before loading onto 3-12% Novex Bis-Tris gradient gels.The electrophoresis was performed in a running buffer containing 50 mMBisTris and 50 mM Tricine (plus 0.004% Coomassie G-250 in cathodebuffer) under fixed voltage (100 V) at 21° C. for 120 min. Proteins weretransferred onto polyvinylidene fluoride membranes and stained withCoomassie G-250 to show molecular-weight markers (NativeMark,Invitrogen). After fixation with 8% acetic acid for 20 min, thepolyvinylidene fluoride membranes were air dried and destained with 100%methanol. Membranes were blocked overnight with 4% BSA in TBS at 4° C.before immunoblotting.

To detected HA tagged BIN2 or GFP tagged bait249, bait249R, bait249NF,bait249NF_Tand on the membrane, the primary antibodies (rat anti-HA,Roche; mouse anti-GFP, Living Colors) have been diluted to 1:1,000 or1:5,000 respectively in PBS containing 0.1% Tween-20 (PBS-T) and 3%(w/v) nonfat milk and incubated for 1 h at RT. After four rinses withPBS-T, the membrane was stained with horseradish peroxidase(HRP)-conjugated goat anti-rat IgG (GE-Healthcare) or sheep anti-mouseIgG (GE-Healthcare) or and visualized with electrochemical luminescencesystem.

7. qRT-PCR

RNA was extracted from whole 8 D.A.S. seedlings treated as indicated in0.5 MS medium and RNA was extracted using the RNeasy kit (Qiagen)according to the supplier's instructions and quantified on a NanoDrop®ND-100 Spectrophotometer. Poly(dT) cDNA was prepared from 1 μg of totalRNA with iScript reverse transcriptase (Biorad). PCR was performed on384-well reaction plates, which were heated for 10 min to 95° C.,followed by 45 cycles of denaturation for 10 s at 95° C. and annealingand extension for 15 s at 60° C. and 72° C., respectively. Targetquantifications were performed with specific primer pairs listed inTable 1. All PCRs were done in three technical repeats, and at least twobiological repeats were used for each sample. For chaperones expressionsanalysis Taqman primer triplets were purchased from Integrated DNATechnologies (IDT). qRT-PCR was performed using the Applied BiosystemsFast Realtime PCR mixture in a Biorad iQ5 machine with detection of theFam fluorophore. Relative expression levels were normalized to CDKA andEF expression levels.

TABLE 1 Primers used for qRT-PCR analysis SEQ Primer ID NO SequenceDWF4FOR 10 GTGATCTCAGCCGTACATTTGGA DWF4REV 11 CACGTCGAAAAACTACCACTTCCTCPDFOR 12 GAATGGAGTGATTACAAGTC CPDREV 13 GTGAACACATTAGAAGGGCCTG NACFOR14 CTCATTTGCCAATCCTGTATC NACREV 15 GCACTGAGATGCGACATCTTG HSP70FOR 16TGACTCTTATCCGCTTGAACAG HSP70REV 17 TCCTACGTTGCTTTCACTGAC HSP90-1FOR 18GTGGTTCCTTCACTGTCACTAG HSP90-1REV 19 TTCACCAAGTCTTTGAGTCTCC HSP101FOR 20TGAAAGGAAGAGGATGCAGC HSP101REV 21 TGTATTTCATCGTGAGAGGCTG HSC70-1FOR 22GCTATTCTCAGCGGTGAAGG HSC70-1REV 23 TTCTCGTCTTGGATGGTGTTC HSC70-2FOR 24GAAACAGAACCACTCCCTCG HSC70-2REV 25 CCAATCAACCTCTTTGCATCG HSC70-3FOR 26AACAGAACCACACCGTCTTAC HSC70-3REV 27 ACCAATCAACCTCTTCGCATC CDKAFOR 28ATTGCGTATTGCCACTCTCATAGG CDKAREV 29 TCCTGACAGGGATACCGAATGC EFFOR 30CTGGAGGTTTTGAGGCTGGTAT EFREV 31 CCAAGGCTGAAAGCAAGAAGA

8. FT-IR Spectroscopy

Fourier Transform Infrared Spectroscopy has been performed on a Tensor37 FT-IR spectrometer equipped with a BioATR II cell (Bruker) aspreviously reported (Xu et al.). Briefly, the detector was cooled withliquid nitrogen, and the Bio-ATR II cell was purged by a continuous flowof dried air to minimize water vapour that may interfere with theresults. Before and after each measurement, the crystal of the ATR cellwas washed with ethanol and water. Samples were measured againstbackground composed of buffer-covered crystal.

1.-15. (canceled)
 16. A method of making a non-naturally occurringmolecule down-regulating the biological function of a protein in a host,the method comprising: i) acquiring the amino acid sequence of saidtarget protein; ii) utilizing a mathematical algorithm to determine anaggregation propensity score and to identify a beta-aggregation regionwithin the amino acid sequence of said target protein; iii) isolatingthe beta-aggregation region from the amino acid encoding sequence ofsaid target protein; and iv) obtain said non-naturally occurringmolecule by fusing the isolated beta-aggregation region to a moiety, toprevent self-aggregation of said beta-aggregation region.
 17. Anon-naturally occurring molecule down-regulating the biological functionof a target protein, comprising a beta-aggregation region fused to amoiety to prevent self-aggregation obtained by the steps comprising: i)acquiring the amino acid sequence of said target protein; ii) utilizinga mathematical algorithm to determine an aggregation propensity scoreand to identify a beta-aggregation region within the amino acid sequenceof said target protein; iii) isolating the beta-aggregation region fromthe amino acid encoding sequence of said target protein; obtain saidnon-naturally occurring molecule by fusing the isolated beta-aggregationregion to a moiety, to prevent self-aggregation of said beta-aggregationregion.
 18. A non-naturally occurring molecule according to claim 17wherein said moiety is a peptide or a protein domain.
 19. Anon-naturally occurring molecule according to claim 17 wherein saidbeta-aggregation region consists of at least 3 contiguous amino acids.20. A non-naturally occurring molecule according to claim 18 wherein apolypeptide linker is present between said beta-aggregation region andsaid moiety.
 21. A non-naturally occurring molecule according to claim18 wherein said peptide or protein domain is not present in the targetprotein.
 22. A recombinant vector comprising a polynucleotide encodingthe molecule of claim 18.